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Mammalian Transcription-Coupled Excision Repair
Vermeulen, Wim; Fousteri, Maria
Transcriptional arrest caused by DNA damage is detrimental for cells and organisms as it impinges on gene expression and thereby on cell growth and survival. To alleviate transcriptional arrest, cells trigger a transcription-dependent genome surveillance pathway, termed transcription-coupled nucleotide excision repair (TC-NER) that ensures rapid removal of such transcription-impeding DNA lesions and prevents persistent stalling of transcription. Defective TC-NER is causatively linked to Cockayne syndrome, a rare severe genetic disorder with multisystem abnormalities that results in patients' death in early adulthood. Here we review recent data on how damage-arrested transcription is actively coupled to TC-NER in mammals and discuss new emerging models concerning the role of TC-NER-specific factors in this process. PMID:23906714
Base excision repair intermediates are mutagenic in mammalian cells
Simonelli, Valeria; Narciso, Laura; Dogliotti, Eugenia; Fortini, Paola
Base excision repair (BER) is the main pathway for repair of DNA damage in mammalian cells. This pathway leads to the formation of DNA repair intermediates which, if still unsolved, cause cell lethality and mutagenesis. To characterize mutations induced by BER intermediates in mammalian cells, an SV-40 derived shuttle vector was constructed carrying a site-specific lesion within the recognition sequence of a restriction endonuclease. The mutation spectra of abasic (AP) sites, 5′-deoxyribose-5-phosphate (5′dRp) and 3′-[2,3-didehydro-2,3-dideoxy-ribose] (3′ddR5p) single-strand breaks (ssb) in mammalian cells was analysed by RFLP/PCR and mutation frequency was estimated by quantitative PCR. Point mutations were the predominant events occurring at all BER intermediates. The AP site-induced mutation spectrum supports evidence for the 'A-rule' and is also consistent with the use of the 5′ neighbouring base to instruct nucleotide incorporation (5′-rule). Preferential adenine insertion was also observed after in vivo replication of 5′dRp or 3′ddR5p ssb. We provide original evidence that not only the abasic site but also its derivatives 'faceless' BER intermediates are mutagenic, with a similar mutation frequency, in mammalian cells. Our findings support the hypothesis that unattended BER intermediates could be a constant threat for genome integrity as well as a spontaneous source of mutations. PMID:16077026
HMGB1 is a cofactor in mammalian base excision repair.
Prasad, Rajendra; Liu, Yuan; Deterding, Leesa J; Poltoratsky, Vladimir P; Kedar, Padmini S; Horton, Julie K; Kanno, Shin-Ichiro; Asagoshi, Kenjiro; Hou, Esther W; Khodyreva, Svetlana N; Lavrik, Olga I; Tomer, Kenneth B; Yasui, Akira; Wilson, Samuel H
Deoxyribose phosphate (dRP) removal by DNA polymerase beta (Pol beta) is a pivotal step in base excision repair (BER). To identify BER cofactors, especially those with dRP lyase activity, we used a Pol beta null cell extract and BER intermediate as bait for sodium borohydride crosslinking. Mass spectrometry identified the high-mobility group box 1 protein (HMGB1) as specifically interacting with the BER intermediate. Purified HMGB1 was found to have weak dRP lyase activity and to stimulate AP endonuclease and FEN1 activities on BER substrates. Coimmunoprecipitation experiments revealed interactions of HMGB1 with known BER enzymes, and GFP-tagged HMGB1 was found to accumulate at sites of oxidative DNA damage in living cells. HMGB1(-/-) mouse cells were slightly more resistant to MMS than wild-type cells, probably due to the production of fewer strand-break BER intermediates. The results suggest HMGB1 is a BER cofactor capable of modulating BER capacity in cells. PMID:17803946
Studying nucleotide excision repair of mammalian DNA in a cell-free system
Wood, R.D.
During nucleotide excision repair, a multiprotein system locates a lesion in DNA and catalyzes enzymatic cleavage of the altered strand. The damaged oligonucleotide and the incision proteins are then displaced, DNA synthesis proceeds to form a short patch using the nonmodified strand as a template, and repair is completed by a DNA ligase. Many gene products participate in these reactions, the best known of which correspond to the seven genetic complementation groups XP-A to XP-G of the disease xeroderma pigmentosum (XP). Cells representing any of these XP groups appear to exhibit, to varying degrees, defects in the first steps of nucleotide excision repair. Individuals affected with XP are hypersensitive to sunlight; most have a predisposition to skin cancer, and some patients show severe neurological abnormalities. In addition to XP, other UV-sensitive mutants of mammalian cells are providing insight into nucleotide excision repair. Of particular interest are mutants isolated from the rodent cells, which have been assigned to 11 different complementation groups. Human genes that can correct the repair defects of rodent mutants in these complementation groups are denoted. ERCC (excision repair cross-complementing) genes are are referred to by number, ERCC1 to ERCC11. Some of these genes are proving to be equivalent to particular XP-complementing genes, while others are distinct. The process of nucleotide excision repair is evolutionarily conserved in eukaryotes, and functional homologues of many of the ERCC and XP genes have been identified in other organisms; studies in yeast are proving to be particularly informative.
Base Excision Repair and Cancer
Wallace, Susan S.; Murphy, Drew L.; Sweasy, Joann B.
Base excision repair is the system used from bacteria to man to remove the tens of thousands of endogenous DNA damages produced daily in each human cell. Base excision repair is required for normal mammalian development and defects have been associated with neurological disorders and cancer. In this paper we provide an overview of short patch base excision repair in humans and summarize current knowledge of defects in base excision repair in mouse models and functional studies on short patch base excision repair germ line polymorphisms and their relationship to cancer. The biallelic germ line mutations that result in MUTYH-associated colon cancer are also discussed. PMID:22252118
In vivo dynamics of chromatin-associated complex formation in mammalian nucleotide excision repair
Moné, Martijn J.; Bernas, Tytus; Dinant, Christoffel; Goedvree, Feliks A.; Manders, Erik M. M.; Volker, Marcel; Houtsmuller, Adriaan B.; Hoeijmakers, Jan H. J.; Vermeulen, Wim; van Driel, Roel
Chromatin is the substrate for many processes in the cell nucleus, including transcription, replication, and various DNA repair systems, all of which require the formation of multiprotein machineries on the chromatin fiber. We have analyzed the kinetics of in vivo assembly of the protein complex that is responsible for nucleotide excision repair (NER) in mammalian cells. Assembly is initiated by UV irradiation of a small area of the cell nucleus, after which the accumulation of GFP-tagged NER proteins in the DNA-damaged area is measured, reflecting the establishment of the dual-incision complex. The dynamic behavior of two NER proteins, ERCC1-XPF and TFIIH, was studied in detail. Results show that the repair complex is assembled with a rate of ≈30 complexes per second and is not diffusion limited. Furthermore, we provide in vivo evidence that not only binding of TFIIH, but also its helicase activity, is required for the recruitment of ERCC1-XPF. These studies give quantitative insight into the de novo assembly of a chromatin-associated protein complex in living cells. PMID:15520397
Molecular mechanisms of DNA damage recognition for mammalian nucleotide excision repair.
Sugasawa, Kaoru
For faithful DNA repair, it is crucial for cells to locate lesions precisely within the vast genome. In the mammalian global genomic nucleotide excision repair (NER) pathway, this difficult task is accomplished through multiple steps, in which the xeroderma pigmentosum group C (XPC) protein complex plays a central role. XPC senses the presence of oscillating 'normal' bases in the DNA duplex, and its binding properties contribute to the extremely broad substrate specificity of NER. Unlike XPC, which acts as a versatile sensor of DNA helical distortion, the UV-damaged DNA-binding protein (UV-DDB) is more specialized, recognizing UV-induced photolesions and facilitating recruitment of XPC. Recent single-molecule analyses and structural studies have advanced our understanding of how UV-DDB finds its targets, particularly in the context of chromatin. After XPC binds DNA, it is necessary to verify the presence of damage in order to avoid potentially deleterious incisions at damage-free sites. Accumulating evidence suggests that XPA and the helicase activity of transcription factor IIH (TFIIH) cooperate to verify abnormalities in DNA chemistry. This chapter reviews recent findings about the mechanisms underlying the efficiency, versatility, and accuracy of NER. PMID:27264556
Adaptive response to gamma radiation in mammalian cells proficient and deficient in components of nucleotide excision repair.
Hafer, Kurt; Iwamoto, Keisuke S; Iwamoto, Keisuke K; Scuric, Zorica; Schiestl, Robert H
Cells preconditioned with low doses of low-linear energy transfer (LET) ionizing radiation become more resistant to later challenges of radiation. The mechanism(s) by which cells adaptively respond to radiation remains unclear, although it has been suggested that DNA repair induced by low doses of radiation increases cellular radioresistance. Recent gene expression profiles have consistently indicated that proteins involved in the nucleotide excision repair pathway are up-regulated after exposure to ionizing radiation. Here we test the role of the nucleotide excision repair pathway for adaptive response to gamma radiation in vitro. Wild-type CHO cells exhibited both greater survival and fewer HPRT mutations when preconditioned with a low dose of gamma rays before exposure to a later challenging dose. Cells mutated for ERCC1, ERCC3, ERCC4 or ERCC5 did not express either adaptive response to radiation; cells mutated for ERCC2 expressed a survival adaptive response but no mutation adaptive response. These results suggest that some components of the nucleotide excision repair pathway are required for phenotypic low-dose induction of resistance to gamma radiation in mammalian cells. PMID:17638404
Genetic evidence for nucleotide excision repair of O6-alkylguanine in mammalian cells.
Boyle, J M; Durrant, L G; Wild, C P; Saffhill, R; Margison, G P
Human cells that lack O6-alkylguanine DNA alkyltransferase (AT) activity can remove O6-butylguanine (O6-nBuG) produced in cellular DNA by exposure to N-n-butyl-N-nitrosourea as determined by radioimmunoassay of enzyme digests of DNA. Fibroblasts from xeroderma pigmentosum (XP) complementation groups A and G that show less than 5% unscheduled DNA synthesis following exposure to UVC failed to remove O6-nBuG. Hence it appears that O6-alkylguanine is repaired in cells that lack AT by a process that is defective in XP cells, presumably nucleotide excision repair. Neither V79 nor V79/79 Chinese hamster cell lines have AT activity and both are able to remove O6-nBuG from DNA. However, only V79/79 is able to remove O6MeG, suggesting some substrate specificity of the excision repair process. Comparison of relative levels of O6-alkylation by N-methyl-, N-ethyl-, N-propyl- and N-n-butyl-nitrosourea indicate that approximately equal levels of O6-alkylation are produced by equitoxic doses of these agents. PMID:3477561
Regulation and Disregulation of Mammalian Nucleotide Excision Repair: a Pathway to Non-germline Breast Carcinogenesis†
Latimer, Jean J.; Majekwana, Vongai J.; Pabón-Padín, Yashira R.; Pimpley, Manasi R.; Grant, Stephen G.
Nucleotide excision repair (NER) is important as a modulator of disease, especially in constitutive deficiencies, such as the cancer predisposition syndrome Xeroderma pigmentosum. We have found profound variation of NER capacity among normal individuals, between cell-types and during carcinogenesis. NER is a repair system for many types of DNA damage, and therefore many types of genotoxic carcinogenic exposures, including ultraviolet light, products of organic combustion, metals, oxidative stress, etc. Since NER is intimately related to cellular metabolism, requiring components of both the DNA replicative and transcription machinery, it has a narrow range of functional viability. Thus, genes in the NER pathway are expressed at the low levels manifested by, for example, nuclear transcription factors. Since NER activity and gene expression vary by cell-type, it is inherently epigenetically regulated. Furthermore, this epigenetic regulation is disregulated during sporadic breast carcinogenesis. Loss of NER is one basis of genomic instability, a required element in cellular transformation, and one that potentially modulates response to therapy. In this paper, we demonstrate differences in NER capacity in eight adult mouse tissues, and place this result into the context of our previous work on mouse extraembryonic tissues, normal human tissues and sporadic early stage human breast cancer. PMID:25393451
Prokaryotic nucleotide excision repair.
Kisker, Caroline; Kuper, Jochen; Van Houten, Bennett
Nucleotide excision repair (NER) has allowed bacteria to flourish in many different niches around the globe that inflict harsh environmental damage to their genetic material. NER is remarkable because of its diverse substrate repertoire, which differs greatly in chemical composition and structure. Recent advances in structural biology and single-molecule studies have given great insight into the structure and function of NER components. This ensemble of proteins orchestrates faithful removal of toxic DNA lesions through a multistep process. The damaged nucleotide is recognized by dynamic probing of the DNA structure that is then verified and marked for dual incisions followed by excision of the damage and surrounding nucleotides. The opposite DNA strand serves as a template for repair, which is completed after resynthesis and ligation. PMID:23457260
Repair of DNA damage in mammalian cells after treatment with UV and dimethyl sulphate: discrimination between nucleotide and base excision repair by their temperature dependence.
Hjertvik, M; Erixon, K; Ahnström, G
Alkylating agents have been reported to give rise to both short and long patches of repair. The reason for the different patch sizes is not known. One possibility is that alkylating agents can trigger both base and nucleotide excision repair. Another possibility is that base excision repair itself can result in different patch sizes. Recognition and incision at lesions is the rate limiting step in excision repair. In order to discriminate between base and nucleotide excision repair it would be desirable to be able to distinguish between different incision activities. In order to accurately measure incision rates, the rejoining of the strand-breaks formed must be inhibited. We have used two inhibitors, aphidicolin and 3-aminobenzamide. Aphidicolin, an inhibitor of DNA polymerases alpha/delta/epsilon. caused accumulation of single-strand breaks both after UV and dimethylsulphate. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose)-polymerase caused accumulation of single-strand breaks only after alkylating agents and is thus specific for base excision repair. Enzymatic activities can be characterised by their activation energy. In order to discriminate between base and nucleotide excision repair the temperature dependence of incision activities was determined. When the temperature is decreased, the incision rate is reduced to a larger extent for UV than for DMS-induced repair. Incisions in UV-irradiated cells are practically cut off at temperatures of 15 degrees C and below, whereas DMS-exposed cells still are actively repairing at this temperature. In DMS treated cells the temperature dependence was the same whether aphidicolin or 3-aminobenzamide was used, speaking against an involvement of nucleotide excision repair. In addition, cell lines deficient in nucleotide excision repair responded in the same way to aphidicolin after DMS treatment as normal cells and were able to make incisions at 15 degrees C. This indicates that nucleotide excision repair is not to any
Nucleotide excision repair in humans.
Spivak, Graciela
The demonstration of DNA damage excision and repair replication by Setlow, Howard-Flanders, Hanawalt and their colleagues in the early 1960s, constituted the discovery of the ubiquitous pathway of nucleotide excision repair (NER). The serial steps in NER are similar in organisms from unicellular bacteria to complex mammals and plants, and involve recognition of lesions, adducts or structures that disrupt the DNA double helix, removal of a short oligonucleotide containing the offending lesion, synthesis of a repair patch copying the opposite undamaged strand, and ligation, to restore the DNA to its original form. The transcription-coupled repair (TCR) subpathway of NER, discovered nearly two decades later, is dedicated to the removal of lesions from the template DNA strands of actively transcribed genes. In this review I will outline the essential factors and complexes involved in NER in humans, and will comment on additional factors and metabolic processes that affect the efficiency of this important process. PMID:26388429
Navigating the Nucleotide Excision Repair Threshold
Liu, Liren; Lee, Jennifer; Zhou, Pengbo
Nucleotide excision repair (NER) is the primary DNA repair pathway that removes helix-distorting DNA strand damage induced by ultraviolet light (UV) irradiation or chemical carcinogens to ensure genome integrity. While the core NER proteins that carry out damage recognition, excision and repair reactions have been identified and extensively characterized, and the NER pathway has been reconstituted in vitro, the regulatory pathways that govern the threshold levels of NER have not been fully elucidated. This mini-review focuses on recently discovered transcriptional and post-translational mechanisms that specify the capacity of NER, and suggests the potential implications of modulating NER activity in cancer prevention and therapeutic intervention. PMID:20458729
Tautomerization-dependent recognition and excision of oxidation damage in base-excision DNA repair.
Zhu, Chenxu; Lu, Lining; Zhang, Jun; Yue, Zongwei; Song, Jinghui; Zong, Shuai; Liu, Menghao; Stovicek, Olivia; Gao, Yi Qin; Yi, Chengqi
NEIL1 (Nei-like 1) is a DNA repair glycosylase guarding the mammalian genome against oxidized DNA bases. As the first enzymes in the base-excision repair pathway, glycosylases must recognize the cognate substrates and catalyze their excision. Here we present crystal structures of human NEIL1 bound to a range of duplex DNA. Together with computational and biochemical analyses, our results suggest that NEIL1 promotes tautomerization of thymine glycol (Tg)-a preferred substrate-for optimal binding in its active site. Moreover, this tautomerization event also facilitates NEIL1-catalyzed Tg excision. To our knowledge, the present example represents the first documented case of enzyme-promoted tautomerization for efficient substrate recognition and catalysis in an enzyme-catalyzed reaction. PMID:27354518
Mammalian DNA Repair. Final Report
The Gordon Research Conference (GRC) on Mammalian DNA Repair was held at Harbortown Resort, Ventura Beach, CA. Emphasis was placed on current unpublished research and discussion of the future target areas in this field.
Differential modulation of base excision repair activities during brain ontogeny: implications for repair of transcribed DNA.
Englander, Ella W; Ma, Huaxian
DNA repair sustains fidelity of genomic replication in proliferating cells and integrity of transcribed sequences in postmitotic tissues. The repair process is critical in the brain, because high oxygen consumption exacerbates the risk for accumulation of oxidative DNA lesions in postmitotic neurons. Most oxidative DNA damage is repaired by the base excision repair (BER) pathway, which is initiated by specialized DNA glycosylases. Because the newly discovered Nei-like mammalian DNA glycosylases (NEIL1/2) proficiently excise oxidized bases from bubble structured DNA, it was suggested that NEILs favor repair of transcribed or replicated DNA. In addition, since NEILs generate 3'-phosphate termini, which are poor targets for AP endonuclease (APE1), it was proposed that APE1-dependent and independent BER sub-pathways exist in mammalian cells. We measured expression and activities of BER enzymes during brain ontogeny, i.e., during a physiologic transition from proliferative to postmitotic differentiated state. While a subset of BER enzymes, exhibited declining expression and excision activities, expression of NEIL1 and NEIL2 glycosylases increased during brain development. Furthermore, the capacity for excision of 5-hydroxyuracil from bubble structured DNA was retained in the mature rat brain suggesting a role for NEIL glycosylases in maintaining the integrity of transcribed DNA in postmitotic brain. PMID:16257035
DNA repair in mammalian embryos.
Jaroudi, Souraya; SenGupta, Sioban
Mammalian cells have developed complex mechanisms to identify DNA damage and activate the required response to maintain genome integrity. Those mechanisms include DNA damage detection, DNA repair, cell cycle arrest and apoptosis which operate together to protect the conceptus from DNA damage originating either in parental gametes or in the embryo's somatic cells. DNA repair in the newly fertilized preimplantation embryo is believed to rely entirely on the oocyte's machinery (mRNAs and proteins deposited and stored prior to ovulation). DNA repair genes have been shown to be expressed in the early stages of mammalian development. The survival of the embryo necessitates that the oocyte be sufficiently equipped with maternal stored products and that embryonic gene expression commences at the correct time. A Medline based literature search was performed using the keywords 'DNA repair' and 'embryo development' or 'gametogenesis' (publication dates between 1995 and 2006). Mammalian studies which investigated gene expression were selected. Further articles were acquired from the citations in the articles obtained from the preliminary Medline search. This paper reviews mammalian DNA repair from gametogenesis to preimplantation embryos to late gestational stages. PMID:17141556
Oxidative DNA Damage and Nucleotide Excision Repair
Melis, Joost P.M.; Luijten, Mirjam
Abstract Significance: Oxidative DNA damage is repaired by multiple, overlapping DNA repair pathways. Accumulating evidence supports the hypothesis that nucleotide excision repair (NER), besides base excision repair (BER), is also involved in neutralizing oxidative DNA damage. Recent Advances: NER includes two distinct sub-pathways: transcription-coupled NER (TC-NER) and global genome repair (GG-NER). The CSA and CSB proteins initiate the onset of TC-NER. Recent findings show that not only CSB, but also CSA is involved in the repair of oxidative DNA lesions, in the nucleus as well as in mitochondria. The XPG protein is also of importance for the removal of oxidative DNA lesions, as it may enhance the initial step of BER. Substantial evidence exists that support a role for XPC in NER and BER. XPC deficiency not only results in decreased repair of oxidative lesions, but has also been linked to disturbed redox homeostasis. Critical Issues: The role of NER proteins in the regulation of the cellular response to oxidative (mitochondrial and nuclear) DNA damage may be the underlying mechanism of the pathology of accelerated aging in Cockayne syndrome patients, a driving force for internal cancer development in XP-A and XP-C patients, and a contributor to the mixed exhibited phenotypes of XP-G patients. Future Directions: Accumulating evidence indicates that DNA repair factors can be involved in multiple DNA repair pathways. However, the distinct detailed mechanism and consequences of these additional functions remain to be elucidated and can possibly shine a light on clinically related issues. Antioxid. Redox Signal. 18, 2409–2419. PMID:23216312
Base Excision Repair in the Mitochondria
Prakash, Aishwarya; Doublié, Sylvie
The 16.5 kb human mitochondrial genome encodes for 13 polypeptides, 22 tRNAs and 2 rRNAs involved in oxidative phosphorylation. Mitochondrial DNA (mtDNA), unlike its nuclear counterpart, is not packaged into nucleosomes and is more prone to the adverse effects of reactive oxygen species (ROS) generated during oxidative phosphorylation. The past few decades have witnessed an increase in the number of proteins observed to translocate to the mitochondria for the purposes of mitochondrial genome maintenance. The mtDNA damage produced by ROS, if not properly repaired, leads to instability and can ultimately manifest in mitochondrial dysfunction and disease. The base excision repair (BER) pathway is employed for the removal and consequently the repair of deaminated, oxidized, and alkylated DNA bases. Specialized enzymes called DNA glycosylases, which locate and cleave the damaged base, catalyze the first step of this highly coordinated repair pathway. This review focuses on members of the four human BER DNA glycosylase superfamilies and their subcellular localization in the mitochondria and/or the nucleus, as well as summarizes their structural features, biochemical properties, and functional role in the excision of damaged bases. PMID:25754732
Base Excision Repair, Aging and Health Span
Xu, Guogang; Herzig, Maryanne; Rotrekl, Vladimir; Walter, Christi A.
DNA damage and mutagenesis are suggested to contribute to aging through their ability to mediate cellular dysfunction. The base excision repair (BER) pathway ameliorates a large number of DNA lesions that arise spontaneously. Many of these lesions are reported to increase with age. Oxidized guanine, repaired largely via base excision repair, is particularly well studied and shown to increase with age. Spontaneous mutant frequencies also increase with age which suggests that mutagenesis may contribute to aging. It is widely accepted that genetic instability contributes to age-related occurrences of cancer and potentially other age-related pathologies. BER activity decreases with age in multiple tissues. The specific BER protein that appears to limit activity varies among tissues. DNA polymerase-β is reduced in brain from aged mice and rats while AP endonuclease is reduced in spermatogenic cells obtained from old mice. The differences in proteins that appear to limit BER activity among tissues may represent true tissue-specific differences in activity or may be due to differences in techniques, environmental conditions or other unidentified differences among the experimental approaches. Much remains to be addressed concerning the potential role of BER in aging and age-related health span. PMID:18423806
Targeting base excision repair for chemosensitization.
Adhikari, Sanjay; Choudhury, Sujata; Mitra, Partha S; Dubash, Jerita J; Sajankila, Shyama P; Roy, Rabindra
In both bacteria and eukaryotes the alkylated, oxidized, and deaminated bases and depurinated lesions are primarily repaired via an endogenous preventive pathway, i.e. base excision repair (BER). Radiation therapy and chemotherapy are two important modes of cancer treatment. Many of those therapeutic agents used in the clinic have the ability to induce the DNA damage; however, they may also be highly cytotoxic, causing peripheral toxicity and secondary cancer as adverse side effects. In addition, the damage produced by the therapeutic agents can often be repaired by the BER proteins, which in effect confers therapeutic resistance. Efficient inhibition of a particular BER protein(s) may increase the efficacy of current chemotherapeutic regimes, which minimizes resistance and ultimately decreases the possibility of the aforementioned negative side effects. Therefore, pharmacological inhibition of DNA damage repair pathways may be explored as a useful strategy to enhance chemosensitivity. Various agents have shown excellent results in preclinical studies in combination chemotherapy. Early phase clinical trials are now being carried out using DNA repair inhibitors targeting enzymes such as PARP, DNA-PK or MGMT. In the case of BER proteins, elimination of N-Methylpurine DNA glycosylase (MPG) or inhibition of AP-endonuclease (APE) increased sensitivity of cancer cells to alkylating chemotherapeutics. MPG(-/-) embryonic stem cells and cells having MPG knock-down by siRNA are hypersensitive to alkylating agents, whereas inhibition of APE by small molecule inhibitors sensitized cancer cells to alkylating chemotherapeutics. Thus, MPG and other BER proteins could be potential targets for chemosensitization. PMID:18473720
Base excision repair capacity in informing healthspan
Brenerman, Boris M.; Illuzzi, Jennifer L.; Wilson, David M.
Base excision repair (BER) is a frontline defense mechanism for dealing with many common forms of endogenous DNA damage, several of which can drive mutagenic or cell death outcomes. The pathway engages proteins such as glycosylases, abasic endonucleases, polymerases and ligases to remove substrate modifications from DNA and restore the genome back to its original state. Inherited mutations in genes related to BER can give rise to disorders involving cancer, immunodeficiency and neurodegeneration. Studies employing genetically defined heterozygous (haploinsufficient) mouse models indicate that partial reduction in BER capacity can increase vulnerability to both spontaneous and exposure-dependent pathologies. In humans, measurement of BER variation has been imperfect to this point, yet tools to assess BER in epidemiological surveys are steadily evolving. We provide herein an overview of the BER pathway and discuss the current efforts toward defining the relationship of BER defects with disease susceptibility. PMID:25355293
Muniandy, Parameswary; Liu, Jia; Majumdar, Alokes; Liu, Su-ting; Seidman, Michael M.
Interstrand DNA crosslinks (ICLs) are formed by natural products of metabolism and by chemotherapeutic reagents. Work in E. coli identified a two cycle repair scheme involving incisions on one strand on either side of the ICL (unhooking) producing a gapped intermediate with the incised oligonucleotide attached to the intact strand. The gap is filled by recombinational repair or lesion bypass synthesis. The remaining monoadduct is then removed by Nucleotide Excision Repair (NER). Despite considerable effort, our understanding of each step in mammalian cells is still quite limited. In part this reflects the variety of crosslinking compounds, each with distinct structural features, used by different investigators. Also, multiple repair pathways are involved, variably operative during the cell cycle. G1 phase repair requires functions from NER, although the mechanism of recognition has not been determined. Repair can be initiated by encounters with the transcriptional apparatus, or a replication fork. In the case of the latter, the reconstruction of a replication fork, stalled or broken by collision with an ICL, adds to the complexity of the repair process. The enzymology of unhooking, the identity of the lesion bypass polymerases required to fill the first repair gap, and the functions involved in the second repair cycle are all subjects of active inquiry. Here we will review current understanding of each step in ICL repair in mammalian cells. PMID:20039786
Base excision repair: A critical player in many games
Wallace, Susan S.
This perspective reviews the many dimensions of base excision repair from a 10,000 foot vantage point and provides one person's view on where the field is headed. Enzyme function is considered under the lens of X-ray diffraction and single molecule studies. Base excision repair in chromatin and telomeres, regulation of expression and the role of posttranslational modifications are also discussed in the context of enzyme activities, cellular localization and interacting partners. The specialized roles that base excision repair play in transcriptional activation by active demethylation and targeted oxidation as well as how base excision repair functions in the immune processes of somatic hypermutation and class switch recombination and its possible involvement in retroviral infection are also discussed. Finally the complexities of oxidative damage and its repair and its link to neurodegenerative disorders, as well as the role of base excision repair as a tumor suppressor are examined in the context of damage, repair and aging. By outlining the many base excision repair-related mysteries that have yet to be unraveled, hopefully this perspective will stimulate further interest in the field. PMID:24780558
Nucleotide excision repair proteins and interstrand crosslink repair
Wood, Richard D.
Although various schemes for interstrand crosslink (ICL) repair incorporate recombination, replication, and double-strand break intermediate steps, action of the NER system or some variation of it is a common feature of most models. In the bacterium Escherichia coli, the NER enzyme UvrABC can incise on either side of an ICL to unhook the crosslink, and repair can proceed via a subsequent recombination step. The relevance of NER to ICL repair in mammalian cells has been challenged. Of all NER mutants, it is clear that ERCC1 and XPF-defective cells show the most pronounced sensitivities to ICL-inducing agents, and defects in ICL repair. However, there is good evidence that cells defective in NER proteins including XPA and XPG are also more sensitive than normal to ICL-inducing agents. These results are summarized here, together with evidence for defective crosslink removal in NER-defective cells. Studies of incision at sites of ICL by cell extracts and purified proteins have been done, but these studies are not all consistent with one another and further research is required. PMID:20658645
Uracil excision repair in Mycobacterium tuberculosis cell-free extracts.
Kumar, Pradeep; Bharti, Sanjay Kumar; Varshney, Umesh
Uracil excision repair is ubiquitous in all domains of life and initiated by uracil DNA glycosylases (UDGs) which excise the promutagenic base, uracil, from DNA to leave behind an abasic site (AP-site). Repair of the resulting AP-sites requires an AP-endonuclease, a DNA polymerase, and a DNA ligase whose combined activities result in either short-patch or long-patch repair. Mycobacterium tuberculosis, the causative agent of tuberculosis, has an increased risk of accumulating uracils because of its G + C-rich genome, and its niche inside host macrophages where it is exposed to reactive nitrogen and oxygen species, two major causes of cytosine deamination (to uracil) in DNA. In vitro assays to study DNA repair in this important human pathogen are limited. To study uracil excision repair in mycobacteria, we have established assay conditions using cell-free extracts of M. tuberculosis and M. smegmatis (a fast-growing mycobacterium) and oligomer or plasmid DNA substrates. We show that in mycobacteria, uracil excision repair is completed primarily via long-patch repair. In addition, we show that M. tuberculosis UdgB, a newly characterized family 5 UDG, substitutes for the highly conserved family 1 UDG, Ung, thereby suggesting that UdgB might function as backup enzyme for uracil excision repair in mycobacteria. PMID:21371942
Variant Base Excision Repair Proteins: Contributors to Genomic Instability
Nemec, Antonia A.; Wallace, Susan S.; Sweasy, Joann B.
Cells sustain endogenous DNA damage at rates greater than 20,000 DNA lesions per cell per day. These damages occur largely as a result of the inherently unstable nature of DNA and the presence of reactive oxygen species within cells. The base excision repair system removes the majority of DNA lesions resulting from endogenous DNA damage. There are several enzymes that function during base excision repair. Importantly, there are over 100 germline single nucleotide polymorphisms in genes that function in base excision repair and that result in non-synonymous amino acid substitutions in the proteins they encode. Somatic variants of these enzymes are also found in human tumors. Variant repair enzymes catalyze aberrant base excision repair. Aberrant base excision repair combined with continuous endogenous DNA damage over time has the potential to lead to a mutator phenotype. Mutations that arise in key growth control genes, imbalances in chromosome number, chromosomal translocations, and loss of heterozygosity can result in the initiation of human cancer or its progression. PMID:20955798
An interplay of the base excision repair and mismatch repair pathways in active DNA demethylation.
Grin, Inga; Ishchenko, Alexander A
Active DNA demethylation (ADDM) in mammals occurs via hydroxylation of 5-methylcytosine (5mC) by TET and/or deamination by AID/APOBEC family enzymes. The resulting 5mC derivatives are removed through the base excision repair (BER) pathway. At present, it is unclear how the cell manages to eliminate closely spaced 5mC residues whilst avoiding generation of toxic BER intermediates and whether alternative DNA repair pathways participate in ADDM. It has been shown that non-canonical DNA mismatch repair (ncMMR) can remove both alkylated and oxidized nucleotides from DNA. Here, a phagemid DNA containing oxidative base lesions and methylated sites are used to examine the involvement of various DNA repair pathways in ADDM in murine and human cell-free extracts. We demonstrate that, in addition to short-patch BER, 5-hydroxymethyluracil and uracil mispaired with guanine can be processed by ncMMR and long-patch BER with concomitant removal of distant 5mC residues. Furthermore, the presence of multiple mispairs in the same MMR nick/mismatch recognition region together with BER-mediated nick formation promotes proficient ncMMR resulting in the reactivation of an epigenetically silenced reporter gene in murine cells. These findings suggest cooperation between BER and ncMMR in the removal of multiple mismatches that might occur in mammalian cells during ADDM. PMID:26843430
An interplay of the base excision repair and mismatch repair pathways in active DNA demethylation
Grin, Inga; Ishchenko, Alexander A.
Active DNA demethylation (ADDM) in mammals occurs via hydroxylation of 5-methylcytosine (5mC) by TET and/or deamination by AID/APOBEC family enzymes. The resulting 5mC derivatives are removed through the base excision repair (BER) pathway. At present, it is unclear how the cell manages to eliminate closely spaced 5mC residues whilst avoiding generation of toxic BER intermediates and whether alternative DNA repair pathways participate in ADDM. It has been shown that non-canonical DNA mismatch repair (ncMMR) can remove both alkylated and oxidized nucleotides from DNA. Here, a phagemid DNA containing oxidative base lesions and methylated sites are used to examine the involvement of various DNA repair pathways in ADDM in murine and human cell-free extracts. We demonstrate that, in addition to short-patch BER, 5-hydroxymethyluracil and uracil mispaired with guanine can be processed by ncMMR and long-patch BER with concomitant removal of distant 5mC residues. Furthermore, the presence of multiple mispairs in the same MMR nick/mismatch recognition region together with BER-mediated nick formation promotes proficient ncMMR resulting in the reactivation of an epigenetically silenced reporter gene in murine cells. These findings suggest cooperation between BER and ncMMR in the removal of multiple mismatches that might occur in mammalian cells during ADDM. PMID:26843430
Rules of Engagement for Base Excision Repair in Chromatin
Odell, Ian D.; Wallace, Susan S.; Pederson, David S.
Most of the DNA in eukaryotes is packaged in tandemly arrayed nucleosomes that, together with numerous DNA- and nucleosome-associated enzymes and regulatory factors, make up chromatin. Chromatin modifying and remodeling agents help regulate access to selected DNA segments in chromatin, thereby facilitating transcription and DNA replication and repair. Studies of nucleotide excision repair (NER), single strand break repair (SSBR), and the homology-directed (HDR) and non-homologous end-joining (NHEJ) double strand break repair pathways have led to an 'access-repair-restore' paradigm, in which chromatin in the vicinity of damaged DNA is disrupted, thereby enabling efficient repair and the subsequent repackaging of DNA into nucleosomes. When damage is extensive, these repair processes are accompanied by cell cycle checkpoint activation, which provides cells with sufficient time to either complete the repair or initiate apoptosis. It is not clear, however, if base excision repair (BER) of the ~20,000 or more oxidative DNA damages that occur daily in each nucleated human cell can be viewed through this same lens. Until recently, we did not know if BER requires or is accompanied by nucleosome disruption, and it is not yet clear that anything short of overwhelming oxidative damage (resulting in the shunting of DNA substrates into other repair pathways) results in checkpoint activation. This review highlights studies of how oxidatively damaged DNA in nucleosomes is discovered and repaired, and offers a working model of events associated with BER in chromatin that we hope will have heuristic value. PMID:22718094
A history of the DNA repair and mutagenesis field: The discovery of base excision repair.
Friedberg, Errol C
This article reviews the early history of the discovery of an DNA repair pathway designated as base excision repair (BER), since in contrast to the enzyme-catalyzed removal of damaged bases from DNA as nucleotides [called nucleotide excision repair (NER)], BER involves the removal of damaged or inappropriate bases, such as the presence of uracil instead of thymine, from DNA as free bases. PMID:26861186
Hydrolytic function of Exo1 in mammalian mismatch repair
Shao, Hongbing; Baitinger, Celia; Soderblom, Erik J.; Burdett, Vickers; Modrich, Paul
Genetic and biochemical studies have previously implicated exonuclease 1 (Exo1) in yeast and mammalian mismatch repair, with results suggesting that function of the protein in the reaction depends on both its hydrolytic activity and its ability to interact with other components of the repair system. However, recent analysis of an Exo1-E109K knockin mouse has concluded that Exo1 function in mammalian mismatch repair is restricted to a structural role, a conclusion based on a prior report that N-terminal His-tagged Exo1-E109K is hydrolytically defective. Because Glu-109 is distant from the nuclease hydrolytic center, we have compared the activity of untagged full-length Exo1-E109K with that of wild type Exo1 and the hydrolytically defective active site mutant Exo1-D173A. We show that the activity of Exo1-E109K is comparable to that of wild type enzyme in a conventional exonuclease assay and that in contrast to a D173A active site mutant, Exo1-E109K is fully functional in mismatch-provoked excision and repair. We conclude that the catalytic function of Exo1 is required for its participation in mismatch repair. We also consider the other phenotypes of the Exo1-E109K mouse in the context of Exo1 hydrolytic function. PMID:24829455
In vitro chromatin templates to study nucleotide excision repair.
Liu, Xiaoqi
In eukaryotic cells, DNA associates with histones and exists in the form of a chromatin hierarchy. Thus, it is generally believed that many eukaryotic cellular DNA processing events such as replication, transcription, recombination and DNA repair are influenced by the packaging of DNA into chromatin. This mini-review covers the current knowledge of DNA damage and repair in chromatin based on in vitro studies. Specifically, nucleosome assembly affects DNA damage formation in both random sequences and sequences with strong nucleosome-positioning signals such as 5S rDNA. At least three systems have been used to analyze the effect of nucleosome folding on nucleotide excision repair (NER) in vitro: (a) human cell extracts that have to rely on labeling of repair synthesis to monitor DNA repair, due to very low repair efficacy; (b) Xenopus oocyte nuclear extracts, that have very robust DNA repair efficacy, have been utilized to follow direct removal of DNA damage; (c) six purified human DNA repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1) that have been used to reconstitute excision repair in vitro. In general, the results have shown that nucleosome folding inhibits NER and, therefore, its activity must be enhanced by chromatin remodeling factors like SWI/SNF. In addition, binding of transcription factors such as TFIIIA to the 5S rDNA promoter also modulates NER efficacy. PMID:26531320
Premature aging and cancer in nucleotide excision repair-disorders
Diderich, K.; Alanazi, M.; Hoeijmakers, J.H.J.
During past decades the major impact of DNA damage on cancer as 'disease of the genes' has become abundantly apparent. In addition to cancer recent years have also uncovered a very strong association of DNA damage with many features of (premature) aging. The notion that DNA repair systems not only protect against cancer but equally against too fast aging has become evident from a systematic, integral analysis of a variety of mouse mutants carrying defects in e.g. transcription-coupled repair with or without an additional impairment of global genome nucleotide excision repair and the corresponding segmental premature aging syndromes in man. A striking correlation between the degree of the DNA repair deficiency and the acceleration of specific progeroid symptoms has been discovered for those repair systems that primarily protect from the cytotoxic and cytostatic effects of DNA damage. These observations are explained from the perspective of nucleotide excision repair mouse mutant and human syndromes. However, similar principles likely apply to other DNA repair pathways including interstrand crosslink repair and double strand break repair and genome maintenance systems in general, supporting the notion that DNA damage constitutes an important intermediate in the process of aging. PMID:21680258
Base excision repair and the role of MUTYH
Kairupan, Carla; Scott, Rodney J
The correction of exogenous and endogenous environmental insult to DNA involves a series of DNA repair mechanisms that reduce the likelihood of mutation accumulation and hence an increased probability of tumour development. The mechanisms underlying the process of base excision repair are relatively well understood and are placed in context with how deterioration of this process is associated with an increased risk of malignancy. PMID:19725997
XPA: A key scaffold for human nucleotide excision repair.
Sugitani, Norie; Sivley, Robert M; Perry, Kelly E; Capra, John A; Chazin, Walter J
Nucleotide excision repair (NER) is essential for removing many types of DNA lesions from the genome, yet the mechanisms of NER in humans remain poorly understood. This review summarizes our current understanding of the structure, biochemistry, interaction partners, mechanisms, and disease-associated mutations of one of the critical NER proteins, XPA. PMID:27247238
Recombinant methods for screening human DNA excision repair proficiency
Athas, W.F.
A method for measuring DNA excision repair in response to ultraviolet radiation (UV)-induced DNA damage has been developed, validated, and field-tested in cultured human lymphocytes. The methodology is amenable to population-based screening and should facilitate future epidemiologic studies seeking to investigate associations between excision repair proficiency and cancer susceptibility. The impetus for such endeavors derives from the belief that the high incidence of skin cancer in the genetic disorder xeroderma pigmentosum (XP) primarily is a result of the reduced capacity of patients cells to repair UV-induced DNA damage. For assay, UV-irradiated non-replicating recombinant plasmid DNA harboring a chloramphenicol acetyltransferase (CAT) indicator gene is introduced into lymphocytes using DEAE-dextran short-term transfection conditions. Exposure to UV induces transcriptionally-inactivating DNA photoproducts in the plasmid DNA which inactivate CAT gene expression. Excision repair of the damaged CAT gene is monitored indirectly as a function of reactivated CAT enzyme activity following a 40 hour repair/expression incubation period.
Complexities of the DNA Base Excision Repair Pathway for Repair of Oxidative DNA Damage
Mitra, Sankar; Boldogh, Istvan; Izumi, Tadahide; Hazra, Tapas K.
Oxidative damage represents the most significant insult to organisms because of continuous production of the reactive oxygen species (ROS) in vivo. Oxidative damage in DNA, a critical target of ROS, is repaired primarily via the base excision repair (BER) pathway which appears to be the simplest among the three excision repair pathways. However, it is now evident that although BER can be carried with four or five enzymes in vitro, a large number of proteins, including some required for nucleotide excision repair (NER), are needed for in vivo repair of oxidative damage. Furthermore, BER in transcribed vs. nontranscribed DNA regions requires distinct sets of proteins, as in the case of NER. We propose an additional complexity in repair of replicating vs. nonreplicating DNA. Unlike DNA bulky adducts, the oxidized base lesions could be incorporated in the nascent DNA strand, repair of which may share components of the mismatch repair process. Distinct enzyme specificities are thus warranted for repair of lesions in the parental vs. nascent DNA strand. Repair synthesis may be carried out by DNA polymerase β or replicative polymerases δ and ε. Thus, multiple subpathways are needed for repairing oxidative DNA damage, and the pathway decision may require coordination of the successive steps in repair. Such coordination includes transfer of the product of a DNA glycosylase to AP-endonuclease, the next enzyme in the pathway. Interactions among proteins in the pathway may also reflect such coordination, characterization of which should help elucidate these subpathways and their in vivo regulation. PMID:11746753
Facilitation of base excision repair by chromatin remodeling.
Hinz, John M; Czaja, Wioletta
Base Excision Repair (BER) is a conserved, intracellular DNA repair system that recognizes and removes chemically modified bases to insure genomic integrity and prevent mutagenesis. Aberrant BER has been tightly linked with a broad spectrum of human pathologies, such as several types of cancer, neurological degeneration, developmental abnormalities, immune dysfunction and aging. In the cell, BER must recognize and remove DNA lesions from the tightly condensed, protein-coated chromatin. Because chromatin is necessarily refractory to DNA metabolic processes, like transcription and replication, the compaction of the genomic material is also inhibitory to the repair systems necessary for its upkeep. Multiple ATP-dependent chromatin remodelling (ACR) complexes play essential roles in modulating the protein-DNA interactions within chromatin, regulating transcription and promoting activities of some DNA repair systems, including double-strand break repair and nucleotide excision repair. However, it remains unclear how BER operates in the context of chromatin, and if the chromatin remodelling processes that govern transcription and replication also actively regulate the efficiency of BER. In this review we highlight the emerging role of ACR in regulation of BER. PMID:26422134
Biomolecular Simulation of Base Excision Repair and Protein Signaling
Straatsma, TP; McCammon, J A; Miller, John H; Smith, Paul E; Vorpagel, Erich R; Wong, Chung F; Zacharias, Martin W
The goal of the Biomolecular Simulation of Base Excision Repair and Protein Signaling project is to enhance our understanding of the mechanism of human polymerase-β, one of the key enzymes in base excision repair (BER) and the cell-signaling enzymes cyclic-AMP-dependent protein kinase. This work used molecular modeling and simulation studies to specifically focus on the • dynamics of DNA and damaged DNA • dynamics and energetics of base flipping in DNA • mechanism and fidelity of nucleotide insertion by BER enzyme human polymerase-β • mechanism and inhibitor design for cyclic-AMP-dependent protein kinase. Molecular dynamics simulations and electronic structure calculations have been performed using the computer resources at the Molecular Science Computing Facility at the Environmental Molecular Sciences Laboratory.
Dynamic control of strand excision during human DNA mismatch repair
Jeon, Yongmoon; Kim, Daehyung; Martín-López, Juana V.; Lee, Ryanggeun; Oh, Jungsic; Hanne, Jeungphill; Fishel, Richard; Lee, Jong-Bong
Mismatch repair (MMR) is activated by evolutionarily conserved MutS homologs (MSH) and MutL homologs (MLH/PMS). MSH recognizes mismatched nucleotides and form extremely stable sliding clamps that may be bound by MLH/PMS to ultimately authorize strand-specific excision starting at a distant 3′- or 5′-DNA scission. The mechanical processes associated with a complete MMR reaction remain enigmatic. The purified human (Homo sapien or Hs) 5′-MMR excision reaction requires the HsMSH2–HsMSH6 heterodimer, the 5′ → 3′ exonuclease HsEXOI, and the single-stranded binding heterotrimer HsRPA. The HsMLH1–HsPMS2 heterodimer substantially influences 5′-MMR excision in cell extracts but is not required in the purified system. Using real-time single-molecule imaging, we show that HsRPA or Escherichia coli EcSSB restricts HsEXOI excision activity on nicked or gapped DNA. HsMSH2–HsMSH6 activates HsEXOI by overcoming HsRPA/EcSSB inhibition and exploits multiple dynamic sliding clamps to increase tract length. Conversely, HsMLH1–HsPMS2 regulates tract length by controlling the number of excision complexes, providing a link to 5′ MMR. PMID:26951673
Repair of radiation damage in mammalian cells
Setlow, R.B.
The responses, such as survival, mutation, and carcinogenesis, of mammalian cells and tissues to radiation are dependent not only on the magnitude of the damage to macromolecular structures - DNA, RNA, protein, and membranes - but on the rates of macromolecular syntheses of cells relative to the half-lives of the damages. Cells possess a number of mechanisms for repairing damage to DNA. If the repair systems are rapid and error free, cells can tolerate much larger doses than if repair is slow or error prone. It is important to understand the effects of radiation and the repair of radiation damage because there exist reasonable amounts of epidemiological data that permits the construction of dose-response curves for humans. The shapes of such curves or the magnitude of the response will depend on repair. Radiation damage is emphasized because: (a) radiation dosimetry, with all its uncertainties for populations, is excellent compared to chemical dosimetry; (b) a number of cancer-prone diseases are known in which there are defects in DNA repair and radiation results in more chromosomal damage in cells from such individuals than in cells from normal individuals; (c) in some cases, specific radiation products in DNA have been correlated with biological effects, and (d) many chemical effects seem to mimic radiation effects. A further reason for emphasizing damage to DNA is the wealth of experimental evidence indicating that damages to DNA can be initiating events in carcinogenesis.
Radiation induced base excision repair (BER): a mechanistic mathematical approach.
Rahmanian, Shirin; Taleei, Reza; Nikjoo, Hooshang
This paper presents a mechanistic model of base excision repair (BER) pathway for the repair of single-stand breaks (SSBs) and oxidized base lesions produced by ionizing radiation (IR). The model is based on law of mass action kinetics to translate the biochemical processes involved, step-by-step, in the BER pathway to translate into mathematical equations. The BER is divided into two subpathways, short-patch repair (SPR) and long-patch repair (LPR). SPR involves in replacement of single nucleotide via Pol β and ligation of the ends via XRCC1 and Ligase III, while LPR involves in replacement of multiple nucleotides via PCNA, Pol δ/ɛ and FEN 1, and ligation via Ligase I. A hallmark of IR is the production of closely spaced lesions within a turn of DNA helix (named complex lesions), which have been attributed to a slower repair process. The model presented considers fast and slow component of BER kinetics by assigning SPR for simple lesions and LPR for complex lesions. In the absence of in vivo reaction rate constants for the BER proteins, we have deduced a set of rate constants based on different published experimental measurements including accumulation kinetics obtained from UVA irradiation, overall SSB repair kinetic experiments, and overall BER kinetics from live-cell imaging experiments. The model was further used to calculate the repair kinetics of complex base lesions via the LPR subpathway and compared to foci kinetic experiments for cells irradiated with γ rays, Si, and Fe ions. The model calculation show good agreement with experimental measurements for both overall repair and repair of complex lesions. Furthermore, using the model we explored different mechanisms responsible for inhibition of repair when higher LET and HZE particles are used and concluded that increasing the damage complexity can inhibit initiation of LPR after the AP site removal step in BER. PMID:25117268
Nucleosomes determine their own patch size in base excision repair
Meas, Rithy; Smerdon, Michael J.
Base excision repair (BER) processes non-helix distorting lesions (e.g., uracils and gaps) and is composed of two subpathways that differ in the number of nucleotides (nts) incorporated during the DNA synthesis step: short patch (SP) repair incorporates 1 nt and long patch (LP) repair incorporates 2–12 nts. This choice for either LP or SP repair has not been analyzed in the context of nucleosomes. Initial studies with uracil located in nucleosome core DNA showed a distinct DNA polymerase extension profile in cell-free extracts that specifically limits extension to 1 nt, suggesting a preference for SP BER. Therefore, we developed an assay to differentiate long and short repair patches in 'designed' nucleosomes containing a single-nucleotide gap at specific locations relative to the dyad center. Using cell-free extracts or purified enzymes, we found that DNA lesions in the nucleosome core are preferentially repaired by DNA polymerase β and there is a significant reduction in BER polymerase extension beyond 1 nt, creating a striking bias for incorporation of short patches into nucleosomal DNA. These results show that nucleosomes control the patch size used by BER. PMID:27265863
Nucleosomes determine their own patch size in base excision repair.
Meas, Rithy; Smerdon, Michael J
Base excision repair (BER) processes non-helix distorting lesions (e.g., uracils and gaps) and is composed of two subpathways that differ in the number of nucleotides (nts) incorporated during the DNA synthesis step: short patch (SP) repair incorporates 1 nt and long patch (LP) repair incorporates 2-12 nts. This choice for either LP or SP repair has not been analyzed in the context of nucleosomes. Initial studies with uracil located in nucleosome core DNA showed a distinct DNA polymerase extension profile in cell-free extracts that specifically limits extension to 1 nt, suggesting a preference for SP BER. Therefore, we developed an assay to differentiate long and short repair patches in 'designed' nucleosomes containing a single-nucleotide gap at specific locations relative to the dyad center. Using cell-free extracts or purified enzymes, we found that DNA lesions in the nucleosome core are preferentially repaired by DNA polymerase β and there is a significant reduction in BER polymerase extension beyond 1 nt, creating a striking bias for incorporation of short patches into nucleosomal DNA. These results show that nucleosomes control the patch size used by BER. PMID:27265863
Effect of the uvrD mutation on excision repair.
Kuemmerle, N B; Masker, W E
A pair of related Escherichia coli K-12 strains, one of which contains the uvrD101 mutation, were constructed and compared for ability to perform various steps in the excision repair of deoxyribonucleic acid damage inflicted by ultraviolet radiation. The results of this study indicated: (i) ultraviolet sensitivity in the uvrD101 mutant was greater than that of wild type but less than that measured in an incision-deficient uvrA mutant; (ii) host cell reactivation paralleled the survival data; (iii) postirradiation deoxyribonucleic acid degradation was virtually identical in the two strains; (iv) incision, presumably at the sites of pyrimidine dimers, proceeded normally in the uvrD101 strain; (v) excision of pyrimidine dimers was markedly reduced in both rate and extent in the uvrD101 mutant; (vi) the amount of repair resynthesis was the same in both strains, and there was no evidence of abnormally long repair patches in the uvrD mutant; and (vii) rejoining of incision breaks was slow and incomplete in the uvrD strain. These data suggest that the ultraviolet sensitivity conferred by the uvrD mutation arises from inefficient removal of pyrimidine dimers or from failure to close incision breaks. The data are compatible with the notion that the uvrD+ gene produce affects the conformation of incised deoxyribonucleic acid molecules. PMID:6991479
Mechanisms of DNA Repair by Photolyase and Excision Nuclease (Nobel Lecture).
Sancar, Aziz
Ultraviolet light damages DNA by converting two adjacent thymines into a thymine dimer which is potentially mutagenic, carcinogenic, or lethal to the organism. This damage is repaired by photolyase and the nucleotide excision repair system in E. coli by nucleotide excision repair in humans. The work leading to these results is presented by Aziz Sancar in his Nobel Lecture. PMID:27337655
Nucleotide excision repair deficient mouse models and neurological disease.
Niedernhofer, Laura J
Nucleotide excision repair (NER) is a highly conserved mechanism to remove helix-distorting DNA base damage. A major substrate for NER is DNA damage caused by environmental genotoxins, most notably ultraviolet radiation. Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy are three human diseases caused by inherited defects in NER. The symptoms and severity of these diseases vary dramatically, ranging from profound developmental delay to cancer predisposition and accelerated aging. All three syndromes include neurological disease, indicating an important role for NER in protecting against spontaneous DNA damage as well. To study the pathophysiology caused by DNA damage, numerous mouse models of NER-deficiency were generated by knocking-out genes required for NER or knocking-in disease-causing human mutations. This review explores the utility of these mouse models to study neurological disease caused by NER-deficiency. PMID:18272436
Properties of damage-dependent DNA incision by nucleotide excision repair in human cell-free extracts.
Calsou, P; Salles, B
Nucleotide excision repair (NER) is the primary mechanism for the removal of many lesions from DNA. This repair process can be broadly divided in two stages: first, incision at damaged sites and second, synthesis of new DNA to replace the oligonucleotide removed by excision. In order to dissect the repair mechanism, we have recently devised a method to analyze the incision reaction in vitro in the absence of repair synthesis (1). Damage-specific incisions take place in a repair reaction in which mammalian cell-free extracts are mixed with undamaged and damaged plasmids. Most of the incision events are accompanied by excision. Using this assay, we investigated here various parameters that specifically affect the level of damage-dependent incision activity by cell-free extracts in vitro. We have defined optimal conditions for the reaction and determined the kinetics of the incision with cell-free extracts from human cells. We present direct evidence that the incision step of NER is ATP-dependent. In addition, we observe that Mn2+ but no other divalent cation can substitute for Mg2+ in the incision reaction. Images PMID:7800483
Excision repair and patch size in UV-irradiated bacteriophage T4
Yarosh, D.B.; Rosenstein, B.S.; Setlow, R.B.
We determined the average size of excision repair patches in repair of UV lesions in bacteriophage T4 by measuring the photolysis of bromodeoxyuridine incorporated during repair. The average patch was small, approximately four nucleotides long. In control experiments with the denV1 excision-deficient mutant, we encountered an artifact, a protein(s) which remained bound to phenol-extracted DNA and prevented nicking by the UV-specific endonucleases of Micrococcus luteus and bacteriophage T4.
Excision repair and patch size in UV-irradiated bacteriophage T4
Yarosh, D.B.; Rosenstein, B.S.; Setlow, R.B.
We determined the average size of excision repair patches in repair of UV lesions in bacteriophage T4 by measuring the photolysis of bromodeoxyuridine incorporated during repair. The average patch was small, approximately four nucleotides long. In control, experiments with the denV/sub 1/ excision-deificient mutant, we encountered an artifact, a protein(s) which remained bound to phenol-extracted DNA and prevented nicking by the UV-specific endonucleases of Micrococcus luteus and bacteriophage T4.
Pol β associated complex and base excision repair factors in mouse fibroblasts.
Prasad, Rajendra; Williams, Jason G; Hou, Esther W; Wilson, Samuel H
During mammalian base excision repair (BER) of lesion-containing DNA, it is proposed that toxic strand-break intermediates generated throughout the pathway are sequestered and passed from one step to the next until repair is complete. This stepwise process is termed substrate channeling. A working model evaluated here is that a complex of BER factors may facilitate the BER process. FLAG-tagged DNA polymerase (pol) β was expressed in mouse fibroblasts carrying a deletion in the endogenous pol β gene, and the cell extract was subjected to an 'affinity-capture' procedure using anti-FLAG antibody. The pol β affinity-capture fraction (ACF) was found to contain several BER factors including polymerase-1, X-ray cross-complementing factor1-DNA ligase III and enzymes involved in processing 3'-blocked ends of BER intermediates, e.g. polynucleotide kinase and tyrosyl-DNA phosphodiesterase 1. In contrast, DNA glycosylases, apurinic/aprymidinic endonuclease 1 and flap endonuclease 1 and several other factors involved in BER were not present. Some of the BER factors in the pol β ACF were in a multi-protein complex as observed by sucrose gradient centrifugation. The pol β ACF was capable of substrate channeling for steps in vitro BER and was proficient in in vitro repair of substrates mimicking a 3'-blocked topoisomerase I covalent intermediate or an oxidative stress-induced 3'-blocked intermediate. PMID:23042675
Polymorphism of the DNA Base Excision Repair Genes in Keratoconus
Wojcik, Katarzyna A.; Synowiec, Ewelina; Sobierajczyk, Katarzyna; Izdebska, Justyna; Blasiak, Janusz; Szaflik, Jerzy; Szaflik, Jacek P.
Keratoconus (KC) is a degenerative corneal disorder for which the exact pathogenesis is not yet known. Oxidative stress is reported to be associated with this disease. The stress may damage corneal biomolecules, including DNA, and such damage is primarily removed by base excision repair (BER). Variation in genes encoding BER components may influence the effectiveness of corneal cells to cope with oxidative stress. In the present work we genotyped 5 polymorphisms of 4 BER genes in 284 patients and 353 controls. The A/A genotype of the c.–1370T>A polymorphism of the DNA polymerase γ (POLG) gene was associated with increased occurrence of KC, while the A/T genotype was associated with decreased occurrence of KC. The A/G genotype and the A allele of the c.1196A>G polymorphism of the X-ray repair cross-complementing group 1 (XRCC1) were associated with increased, and the G/G genotype and the G allele, with decreased KC occurrence. Also, the C/T and T as well as C/C genotypes and alleles of the c.580C>T polymorphism of the same gene displayed relationship with KC occurrence. Neither the g.46438521G>C polymorphism of the Nei endonuclease VIII-like 1 (NEIL1) nor the c.2285T>C polymorphism of the poly(ADP-ribose) polymerase-1 (PARP-1) was associated with KC. In conclusion, the variability of the XRCC1 and POLG genes may play a role in KC pathogenesis and determine the risk of this disease. PMID:25356504
Mismatch repair and nucleotide excision repair proteins cooperate in the recognition of DNA interstrand crosslinks.
Zhao, Junhua; Jain, Aklank; Iyer, Ravi R; Modrich, Paul L; Vasquez, Karen M
DNA interstrand crosslinks (ICLs) are among the most cytotoxic types of DNA damage, thus ICL-inducing agents such as psoralen, are clinically useful chemotherapeutics. Psoralen-modified triplex-forming oligonucleotides (TFOs) have been used to target ICLs to specific genomic sites to increase the selectivity of these agents. However, how TFO-directed psoralen ICLs (Tdp-ICLs) are recognized and processed in human cells is unclear. Previously, we reported that two essential nucleotide excision repair (NER) protein complexes, XPA-RPA and XPC-RAD23B, recognized ICLs in vitro, and that cells deficient in the DNA mismatch repair (MMR) complex MutSbeta were sensitive to psoralen ICLs. To further investigate the role of MutSbeta in ICL repair and the potential interaction between proteins from the MMR and NER pathways on these lesions, we performed electrophoretic mobility-shift assays and chromatin immunoprecipitation analysis of MutSbeta and NER proteins with Tdp-ICLs. We found that MutSbeta bound to Tdp-ICLs with high affinity and specificity in vitro and in vivo, and that MutSbeta interacted with XPA-RPA or XPC-RAD23B in recognizing Tdp-ICLs. These data suggest that proteins from the MMR and NER pathways interact in the recognition of ICLs, and provide a mechanistic link by which proteins from multiple repair pathways contribute to ICL repair. PMID:19468048
The Fanconi Anaemia Components UBE2T and FANCM Are Functionally Linked to Nucleotide Excision Repair
Kelsall, Ian R.; Langenick, Judith; MacKay, Craig; Patel, Ketan J.; Alpi, Arno F.
The many proteins that function in the Fanconi anaemia (FA) monoubiquitylation pathway initiate replicative DNA crosslink repair. However, it is not clear whether individual FA genes participate in DNA repair pathways other than homologous recombination and translesion bypass. Here we show that avian DT40 cell knockouts of two integral FA genes – UBE2T and FANCM are unexpectedly sensitive to UV-induced DNA damage. Comprehensive genetic dissection experiments indicate that both of these FA genes collaborate to promote nucleotide excision repair rather than translesion bypass to protect cells form UV genotoxicity. Furthermore, UBE2T deficiency impacts on the efficient removal of the UV-induced photolesion cyclobutane pyrimidine dimer. Therefore, this work reveals that the FA pathway shares two components with nucleotide excision repair, intimating not only crosstalk between the two major repair pathways, but also potentially identifying a UBE2T-mediated ubiquitin-signalling response pathway that contributes to nucleotide excision repair. PMID:22615860
Polymorphisms of nucleotide excision repair genes predict melanoma survival.
Li, Chunying; Yin, Ming; Wang, Li-E; Amos, Christopher I; Zhu, Dakai; Lee, Jeffrey E; Gershenwald, Jeffrey E; Grimm, Elizabeth A; Wei, Qingyi
Melanoma is the most highly malignant skin cancer, and nucleotide excision repair (NER) is involved in melanoma susceptibility. In this analysis of 1,042 melanoma patients, we evaluated whether genetic variants of NER genes may predict survival outcome of melanoma patients. We used genotyping data of 74 tagging single-nucleotide polymorphisms (tagSNPs) in eight core NER genes from our genome-wide association study (including two in XPA, 14 in XPC, three in XPE, four in ERCC1, 10 in ERCC2, eight in ERCC3, 14 in ERCC4, and 19 in ERCC5) and evaluated their associations with prognosis of melanoma patients. Using the Cox proportional hazards model and Kaplan-Meier analysis, we found a predictive role of XPE rs28720291, ERCC5 rs4150314, XPC rs2470458, and ERCC2 rs50871 SNPs in the prognosis of melanoma patients (rs28720291: AG vs. GG, adjusted hazard ratio (adjHR)=11.2, 95% confidence interval (CI) 3.04-40.9, P=0.0003; rs4150314: AG vs. GG, adjHR=4.76, 95% CI 1.09-20.8, P=0.038; rs2470458: AA vs. AG/GG, adjHR=2.11, 95% CI 1.03-4.33, P=0.040; and rs50871: AA vs. AC/CC adjHR=2.27, 95% CI 1.18-4.35, P=0.015). Patients with an increasing number of unfavorable genotypes had markedly increased death risk. Genetic variants of NER genes, particularly XPE rs28720291, ERCC5 rs4150314, XPC rs2470458, and ERCC2 rs50871, may independently or jointly modulate survival outcome of melanoma patients. Because our results were based on a median follow-up of 3 years without multiple test corrections, additional large prospective studies are needed to confirm our findings. PMID:23407396
DNA repair and radiation sensitivity in mammalian cells
Chen, D.J.C.; Stackhouse, M. ); Chen, D.S. . Dept. of Radiation Oncology)
Ionizing radiation induces various types of damage in mammalian cells including DNA single-strand breaks, DNA double-strand breaks (DSB), DNA-protein cross links, and altered DNA bases. Although human cells can repair many of these lesions there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. We report here on the cellular and genetic analyses of DNA double-strand break repair deficient mammalian cells. It has been well established that the DNA double-strand break is one of the major lesions induced by ionizing radiation. Utilizing rodent repair-deficient mutant, we have shown that the genes responsible for DNA double-strand break repair are also responsible for the cellular expression of radiation sensitivity. The molecular genetic analysis of DSB repair in rodent/human hybrid cells indicate that at least 6 different genes in mammalian cells are responsible for the repair of radiation-induced DNA double-strand breaks. Mapping and the prospect of cloning of human radiation repair genes are reviewed. Understanding the molecular and genetic basis of radiation sensitivity and DNA repair in man will provide a rational foundation to predict the individual risk associated with radiation exposure and to prevent radiation-induced genetic damage in the human population.
DNA repair and radiation sensitivity in mammalian cells
Chen, D.J.C.; Stackhouse, M.; Chen, D.S.
Ionizing radiation induces various types of damage in mammalian cells including DNA single-strand breaks, DNA double-strand breaks (DSB), DNA-protein cross links, and altered DNA bases. Although human cells can repair many of these lesions there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. We report here on the cellular and genetic analyses of DNA double-strand break repair deficient mammalian cells. It has been well established that the DNA double-strand break is one of the major lesions induced by ionizing radiation. Utilizing rodent repair-deficient mutant, we have shown that the genes responsible for DNA double-strand break repair are also responsible for the cellular expression of radiation sensitivity. The molecular genetic analysis of DSB repair in rodent/human hybrid cells indicate that at least 6 different genes in mammalian cells are responsible for the repair of radiation-induced DNA double-strand breaks. Mapping and the prospect of cloning of human radiation repair genes are reviewed. Understanding the molecular and genetic basis of radiation sensitivity and DNA repair in man will provide a rational foundation to predict the individual risk associated with radiation exposure and to prevent radiation-induced genetic damage in the human population.
Base Excision Repair Facilitates a Functional Relationship Between Guanine Oxidation and Histone Demethylation
Li, Jianfeng; Braganza, Andrea
Abstract Significance: Appropriately controlled epigenetic regulation is critical for the normal development and health of an organism. Misregulation of epigenetic control via deoxyribonucleic acid (DNA) methylation or histone methylation has been associated with cancer and chromosomal instability syndromes. Recent Advances: The main function of the proteins in the base excision repair (BER) pathway is to repair DNA single-strand breaks and deamination, oxidation, and alkylation-induced DNA base damage that may result from chemotherapy, environmental exposure, or byproducts of cellular metabolism. Recent studies have suggested that one or more BER proteins may also participate in epigenetic regulation to facilitate gene expression modulation via alteration of the state of DNA methylation or via a reaction coupled to histone modification. BER proteins have also been reported to play an essential role in pluripotent stem cell reprogramming. Critical Issues: One emerging function for BER in epigenetic regulation is the repair of base lesions induced by hydrogen peroxide as a byproduct of lysine-specific demethylase 1 (LSD1) enzymatic activity (LSD1/LSD2-coupled BER) for transcriptional regulation. Future Directions: To shed light on this novel role of BER, this review focuses on the repair of oxidative lesions in nuclear DNA that are induced during LSD1-mediated histone demethylation. Further, we highlight current studies suggesting a role for BER proteins in transcriptional regulation of gene expression via BER-coupled active DNA demethylation in mammalian cells. Such efforts to address the role of BER proteins in epigenetic regulation could broaden cancer therapeutic strategies to include epigenetic modifiers combined with BER inhibitors. Antioxid. Redox Signal. 18, 2429–2443. PMID:23311711
SUMOylation of xeroderma pigmentosum group C protein regulates DNA damage recognition during nucleotide excision repair
Akita, Masaki; Tak, Yon-Soo; Shimura, Tsutomu; Matsumoto, Syota; Okuda-Shimizu, Yuki; Shimizu, Yuichiro; Nishi, Ryotaro; Saitoh, Hisato; Iwai, Shigenori; Mori, Toshio; Ikura, Tsuyoshi; Sakai, Wataru; Hanaoka, Fumio; Sugasawa, Kaoru
The xeroderma pigmentosum group C (XPC) protein complex is a key factor that detects DNA damage and initiates nucleotide excision repair (NER) in mammalian cells. Although biochemical and structural studies have elucidated the interaction of XPC with damaged DNA, the mechanism of its regulation in vivo remains to be understood in more details. Here, we show that the XPC protein undergoes modification by small ubiquitin-related modifier (SUMO) proteins and the lack of this modification compromises the repair of UV-induced DNA photolesions. In the absence of SUMOylation, XPC is normally recruited to the sites with photolesions, but then immobilized profoundly by the UV-damaged DNA-binding protein (UV-DDB) complex. Since the absence of UV-DDB alleviates the NER defect caused by impaired SUMOylation of XPC, we propose that this modification is critical for functional interactions of XPC with UV-DDB, which facilitate the efficient damage handover between the two damage recognition factors and subsequent initiation of NER. PMID:26042670
Human HMGB1 directly facilitates interactions between nucleotide excision repair proteins on triplex-directed psoralen interstrand crosslinks
Lange, Sabine S.; Reddy, Madhava C.; Vasquez, Karen M.
Psoralen is a chemotherapeutic agent that acts by producing DNA interstrand crosslinks (ICLs), which are especially cytotoxic and mutagenic because their complex chemical nature makes them difficult to repair. Proteins from multiple repair pathways, including nucleotide excision repair (NER), are involved in their removal in mammalian cells, but the exact nature of their repair is poorly understood. We have shown previously that HMGB1, a protein involved in chromatin structure, transcriptional regulation, and inflammation, can bind cooperatively to triplex-directed psoralen ICLs with RPA, and that mammalian cells lacking HMGB1 are hyper-sensitive to psoralen ICLs. However, whether this effect is mediated by a role for HMGB1 in DNA damage recognition is still unknown. Given HMGB1's ability to bind to damaged DNA and its interaction with the RPA protein, we hypothesized that HMGB1 works together with the NER damage recognition proteins to aid in the removal of ICLs. We show here that HMGB1 is capable of binding to triplex-directed psoralen ICLs with the dedicated NER damage recognition complex XPC-RAD23B, as well as RPA, and that they form a high molecular weight complex on these lesions. In addition, we demonstrate that HMGB1 interacts with XPC-RAD23B and XPA in the absence of DNA. These findings directly demonstrate interactions between HMGB1 and the NER damage recognition proteins, and suggest that HMGB1 may affect ICL repair by enhancing the interactions between NER damage recognition factors. PMID:19446504
Reprint of "Oxidant and environmental toxicant-induced effects compromise DNA ligation during base excision DNA repair".
Çağlayan, Melike; Wilson, Samuel H
DNA lesions arise from many endogenous and environmental agents, and such lesions can promote deleterious events leading to genomic instability and cell death. Base excision repair (BER) is the main DNA repair pathway responsible for repairing single strand breaks, base lesions and abasic sites in mammalian cells. During BER, DNA substrates and repair intermediates are channeled from one step to the next in a sequential fashion so that release of toxic repair intermediates is minimized. This includes handoff of the product of gap-filling DNA synthesis to the DNA ligation step. The conformational differences in DNA polymerase β (pol β) associated with incorrect or oxidized nucleotide (8-oxodGMP) insertion could impact channeling of the repair intermediate to the final step of BER, i.e., DNA ligation by DNA ligase I or the DNA Ligase III/XRCC1 complex. Thus, modified DNA ligase substrates produced by faulty pol β gap-filling could impair coordination between pol β and DNA ligase. Ligation failure is associated with 5'-AMP addition to the repair intermediate and accumulation of strand breaks that could be more toxic than the initial DNA lesions. Here, we provide an overview of the consequences of ligation failure in the last step of BER. We also discuss DNA-end processing mechanisms that could play roles in reversal of impaired BER. PMID:26596511
Activities of DNA base excision repair enzymes in liver and brain correlate with body mass, but not lifespan.
Page, Melissa M; Stuart, Jeffrey A
Accumulation of DNA lesions compromises replication and transcription and is thus toxic to cells. DNA repair deficiencies are generally associated with cellular replicative senescence and premature aging syndromes, suggesting that efficient DNA repair is required for normal longevity. It follows that the evolution of increasing lifespan amongst animal species should be associated with enhanced DNA repair capacities. Although UV damage repair has been shown to correlate positively with mammalian species lifespan, we lack similar insight into many other DNA repair pathways, including base excision repair (BER). DNA is continuously exposed to reactive oxygen species produced during aerobic metabolism, resulting in the occurrence of oxidative damage within DNA. Short-patch BER plays an important role in repairing the resultant oxidative lesions. We therefore tested whether an enhancement of BER enzyme activities has occurred concomitantly with the evolution of increased maximum lifespan (MLSP). We collected brain and liver tissue from 15 vertebrate endotherm species ranging in MLSP over an order of magnitude. We measured apurinic/apyrimidinic (AP) endonuclease activity, as well as the rates of nucleotide incorporation into an oligonucleotide containing a single nucleotide gap (catalyzed by BER polymerase β) and subsequent ligation of the oligonucleotide. None of these activities correlated positively with species MLSP. Rather, nucleotide incorporation and oligonucleotide ligation activities appeared to be primarily (and negatively) correlated with species body mass. PMID:21853261
Postreplication repair in mammalian cells after ultraviolet irradiation: a model.
Lavin, M F
A model is presented for bypass of ultraviolet-induced damage in DNA during replication. The overall process is initiated by the introduction of a single-strand break into parental DNA near the point of arrest of synthesis, followed by a transient crossing-over step similar to that envisaged in genetic recombination. The mechanism proposed provides an alternative explanation to existing models and is entirely consistent with available data on postreplication repair in mammalian cells. In addition the model explains the low level of recombination repair observed in mammalian cells. PMID:687763
Repair of mismatched basepairs in mammalian DNA
Taylor, J.H.; Hare, J.T.
We have concentrated on three specific areas of our research plan. Our greatest emphasis is on the role of single strand nicks in influencing template strand selection in mismatch repair. We have found, that the ability of a nick in one strand to influence which strand is repaired is not a simple function of distance from the mismatched site but rather that an hot spot where a nick is more likely to have an influence can exist. The second line was production of single-genotype heteroduplexes in order to examine independently the repair of T/G and A/C mispairs within the same sequence context as in our mixed mispair preparations. We have shown preparations of supercoiled heteroduplex can be prepared that were exclusively T/G or exclusively A/C at the mispair site. The third effort has been to understand the difference in repair bias of different cell lines or different transfection conditions as it may relate to different repair systems in the cell. We have identified some of the sources of variation, including cell cycle position. We hope to continue this work to more precisely identify the phase of the cell cycle.
Inhibition of excision-repair of ultraviolet damage in human cells by exposure to methyl methanesulfonate.
Park, S D; Choi, K H; Hong, S W; Cleaver, J E
Unscheduled DNA synthesis and excision of pyrimidine dimers in human cells exposed to ultraviolet let were inhibited by exposure to methyl methanesulfonate (MMS, 1-2 mM), but repair of MMS damage was not inhibited by UV light. Because the pathways for excision of pyrimidine dimers and alkylation damage have previously been shown to be different, this observation implies a direct effect of alkylation on repair enzymes. We estimate that if inhibition is due to protein alkylation, the UV repair system must present an extremely large target to alkylation and may involve a complex of protein subunits in the order of 1 million daltons such that 1 or more alkylations occur per complex at the concentrations used. These results also indicate that the method of exposing cells to 2 DNA-damaging agents to determine whether they are repaired by common or different pathways can be quite unreliable because of other effects on the repair systems themselves. PMID:7196494
The ING1b tumor suppressor facilitates nucleotide excision repair by promoting chromatin accessibility to XPA
Kuo, Wei-Hung W.; Wang Yemin; Wong, Ronald P.C.; Campos, Eric I.; Li Gang . E-mail: firstname.lastname@example.com
ING1b is the most studied ING family protein and perhaps the most ubiquitously and abundantly expressed. This protein is involved in the regulation of various biological functions ranging from senescence, cell cycle arrest, apoptosis, to DNA repair. ING1b is upregulated by UV irradiation and enhances the removal of bulky nucleic acid photoproducts. In this study, we provide evidence that ING1b mediates nucleotide excision repair by facilitating the access to damaged nucleosomal DNA. We demonstrate that ING1b is not recruited to UV-induced DNA lesions but enhances nucleotide excision repair only in XPC-proficient cells, implying an essential role in early steps of the 'access, repair, restore' model. We also find that ING1b alters histone acetylation dynamics upon exposure to UV radiation and induces chromatin relaxation in microccocal nuclease digestion assay, revealing that ING1b may allow better access to nucleotide excision repair machinery. More importantly, ING1b associates with chromatin in a UV-inducible manner and facilitates DNA access to nucleotide excision repair factor XPA. Furthermore, depletion of the endogenous ING1b results to the sensitization of cells at S-phase to UV irradiation. Taken together, these observations establish a role of ING1b acting as a chromatin accessibility factor for DNA damage recognition proteins upon genotoxic injury.
Quantitative characterization of protein–protein complexes involved in base excision DNA repair
Moor, Nina A.; Vasil'eva, Inna A.; Anarbaev, Rashid O.; Antson, Alfred A.; Lavrik, Olga I.
Base Excision Repair (BER) efficiently corrects the most common types of DNA damage in mammalian cells. Step-by-step coordination of BER is facilitated by multiple interactions between enzymes and accessory proteins involved. Here we characterize quantitatively a number of complexes formed by DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), poly(ADP-ribose) polymerase 1 (PARP1), X-ray repair cross-complementing protein 1 (XRCC1) and tyrosyl-DNA phosphodiesterase 1 (TDP1), using fluorescence- and light scattering-based techniques. Direct physical interactions between the APE1-Polβ, APE1-TDP1, APE1-PARP1 and Polβ-TDP1 pairs have been detected and characterized for the first time. The combined results provide strong evidence that the most stable complex is formed between XRCC1 and Polβ. Model DNA intermediates of BER are shown to induce significant rearrangement of the Polβ complexes with XRCC1 and PARP1, while having no detectable influence on the protein–protein binding affinities. The strength of APE1 interaction with Polβ, XRCC1 and PARP1 is revealed to be modulated by BER intermediates to different extents, depending on the type of DNA damage. The affinity of APE1 for Polβ is higher in the complex with abasic site-containing DNA than after the APE1-catalyzed incision. Our findings advance understanding of the molecular mechanisms underlying coordination and regulation of the BER process. PMID:26013813
Subunit interactions in yeast transcription/repair factor TFIIH. Requirement for Tfb3 subunit in nucleotide excision repair.
Feaver, W J; Huang, W; Gileadi, O; Myers, L; Gustafsson, C M; Kornberg, R D; Friedberg, E C
A yeast strain harboring a temperature-sensitive allele of TFB3 (tfb3(ts)), the 38-kDa subunit of the RNA polymerase II transcription/nucleotide excision repair factor TFIIH, was found to be sensitive to ultraviolet (UV) radiation and defective for nucleotide excision repair in vitro. Interestingly, tfb3(ts) failed to grow on medium containing caffeine. A comprehensive pairwise two-hybrid analysis between yeast TFIIH subunits identified novel interactions between Rad3 and Tfb3, Tfb4 and Ssl1, as well as Ssl2 and Tfb2. These interactions have facilitated a more complete model of the structure of TFIIH and the nucleotide excision repairosome. PMID:10681587
Purification of mammalian DNA repair protein XRCC1
Chen, I.
Malfunctioning DNA repair systems lead to cancer mutations, and cell death. XRCC1 (X-ray Repair Cross Complementing) is a human DNA repair gene that has been found to fully correct the x-ray repair defect in Chinese hamster ovary (CHO) cell mutant EM9. The corresponding protein (XRCC1) encoded by this gene has been linked to a DNA repair pathway known as base excision repair, and affects the activity of DNA ligase III. Previously, an XRCC1 cDNA minigene (consisting of the uninterrupted coding sequence for XRCC1 protein followed by a decahistidine tag) was constructed and cloned into vector pET-16b for the purpose of: (1) overproduction of XRCC1 in both prokaryotic and eukaryotic cells; and (2) to facilitate rapid purification of XRCC1 from these systems. A vector is basically a DNA carrier that allows recombinant protein to be cloned and overexpressed in host cells. In this study, XRCC1 protein was overexpressed in E. coli and purified by immobilized metal affinity chromatography. Currently, the XRCC1 minigene is being inserted into a new vector [pET-26b(+)] in hopes to increase overexpression and improve purification. Once purified XRCC1 can be crystallized for structural studies, or studied in vitro for its biological function.
Induced DNA repair pathway in mammalian cells
Overberg, R.
The survival of cultured rat kangaroo cells (PtK-2) and human xeroderma pigmentosum cells incubated with 5 ..mu..M cycloheximide subsequent to ultraviolet irradiation is lower than that of cells incubated without cycloheximide. The drop in survival is considerably larger than that produced by incubation of unirradiated cells with cycloheximide. The phenomenon was also observed when PtK-2 cells were incubated with emetine, another protein synthesis inhibitor, or with 5,6-dichloro-1-..beta..-D-ribofuranosylbenzimidazole, a RNA synthesis inhibitor. PtK cells which received a preliminary UV treatment followed by an incubation period without cycloheximide and then a second irradiation and 24 hour incubation with cycloheximide, survived the effects of the second irradiation better than cells which were incubated in the presence of cycloheximide after the first and second UV irradiation. The application of cycloheximide for 24 hours after UV irradiation of PtK cells resulted in one-half as many 6-thioguanine resistant cells as compared to the number of 6-thioguanine resistant cells found when cycloheximide was not used. These experiments indicate that a UV-inducible cycloheximide-sensitive DNA repair pathway is present in PtK and xeroderma pigmentosum cells, which is error-prone in PtK cells.
The lung cancer mortality rate in Xuan Wei County, China is among the highest in the country and has been etiologically attributed to exposure to indoor smoky coal emissions that contain very high levels of polycyclic aromatic hydrocarbons (PAHs). Nucleotide excision repair (NE...
Excision repair of UV radiation-induced DNA damage in Caenorhabditis elegans
Hartman, P.S.; Hevelone, J.; Dwarakanath, V.; Mitchell, D.L. )
Radioimmunoassays were used to monitor the removal of antibody-binding sites associated with the two major UV radiation-induced DNA photoproducts (cyclobutane dimers and (6-4) photoproducts). Unlike with cultured human cells, where (6-4) photoproducts are removed more rapidly than cyclobutane dimers, the kinetics of repair were similar for both lesions. Repair capacity in wild type diminished throughout development. The radioimmunoassays were also employed to confirm the absence of photoreactivation in C. elegans. In addition, three radiation-sensitive mutants (rad-1, rad-2, rad-7) displayed normal repair capacities. An excision defect was much more pronounced in larvae than embryos in the fourth mutant tested (rad-3). This correlates with the hypersensitivity pattern of this mutant and suggests that DNA repair may be developmentally regulated in C. elegans. The mechanism of DNA repair in C. elegans as well as the relationship between the repair of specific photoproducts and UV radiation sensitivity during development are discussed.
Nucleotide excision repair in rat male germ cells: low level of repair in intact cells contrasts with high dual incision activity in vitro.
Jansen, J; Olsen, A K; Wiger, R; Naegeli, H; de Boer, P; van Der Hoeven, F; Holme, J A; Brunborg, G; Mullenders, L
The acquisition of genotoxin-induced mutations in the mammalian germline is detrimental to the stable transfer of genomic information. In somatic cells, nucleotide excision repair (NER) is a major pathway to counteract the mutagenic effects of DNA damage. Two NER subpathways have been identified, global genome repair (GGR) and transcription-coupled repair (TCR). In contrast to somatic cells, little is known regarding the expression of these pathways in germ cells. To address this basic question, we have studied NER in rat spermatogenic cells in crude cell suspension, in enriched cell stages and within seminiferous tubules after exposure to UV or N-acetoxy-2-acetylaminofluorene. Surprisingly, repair in spermatogenic cells was inefficient in the genome overall and in transcriptionally active genes indicating non-functional GGR and TCR. In contrast, extracts from early/mid pachytene cells displayed dual incision activity in vitro as high as extracts from somatic cells, demonstrating that the proteins involved in incision are present and functional in premeiotic cells. However, incision activities of extracts from diplotene cells and round spermatids were low, indicating a stage-dependent expression of incision activity. We hypothesize that sequestering of NER proteins by mispaired regions in DNA involved in synapsis and recombination may underlie the lack of NER activity in premeiotic cells. PMID:11292852
Low-Dose Formaldehyde Delays DNA Damage Recognition and DNA Excision Repair in Human Cells
Luch, Andreas; Frey, Flurina C. Clement; Meier, Regula; Fei, Jia; Naegeli, Hanspeter
Objective Formaldehyde is still widely employed as a universal crosslinking agent, preservative and disinfectant, despite its proven carcinogenicity in occupationally exposed workers. Therefore, it is of paramount importance to understand the possible impact of low-dose formaldehyde exposures in the general population. Due to the concomitant occurrence of multiple indoor and outdoor toxicants, we tested how formaldehyde, at micromolar concentrations, interferes with general DNA damage recognition and excision processes that remove some of the most frequently inflicted DNA lesions. Methodology/Principal Findings The overall mobility of the DNA damage sensors UV-DDB (ultraviolet-damaged DNA-binding) and XPC (xeroderma pigmentosum group C) was analyzed by assessing real-time protein dynamics in the nucleus of cultured human cells exposed to non-cytotoxic (<100 μM) formaldehyde concentrations. The DNA lesion-specific recruitment of these damage sensors was tested by monitoring their accumulation at local irradiation spots. DNA repair activity was determined in host-cell reactivation assays and, more directly, by measuring the excision of DNA lesions from chromosomes. Taken together, these assays demonstrated that formaldehyde obstructs the rapid nuclear trafficking of DNA damage sensors and, consequently, slows down their relocation to DNA damage sites thus delaying the excision repair of target lesions. A concentration-dependent effect relationship established a threshold concentration of as low as 25 micromolar for the inhibition of DNA excision repair. Conclusions/Significance A main implication of the retarded repair activity is that low-dose formaldehyde may exert an adjuvant role in carcinogenesis by impeding the excision of multiple mutagenic base lesions. In view of this generally disruptive effect on DNA repair, we propose that formaldehyde exposures in the general population should be further decreased to help reducing cancer risks. PMID:24722772
Modeling the induced mutation process in bacterial cells with defects in excision repair system
NASA Astrophysics Data System (ADS)
Bugay, A. N.; Vasilyeva, M. A.; Krasavin, E. A.; Parkhomenko, A. Yu.
A mathematical model of the UV-induced mutation process in Escherichia coli cells with defects in the uvrA and polA genes has been developed. The model describes in detail the reaction kinetics for the excision repair system. The number of mismatches as a result of translesion synthesis is calculated for both wild-type and mutant cells. The effect of temporal modulation of the number of single-stranded DNA during postreplication repair has been predicted. A comparison of effectiveness of different repair systems has been conducted.
Nucleotide excision repair and response and survival to chemotherapy in colorectal cancer patients.
Kap, Elisabeth J; Popanda, Odilia; Chang-Claude, Jenny
Several new chemotherapeutic agents have become available for the treatment of colorectal cancer, which has led to increased complexity in treatment planning. Treatment decision making for individual patients could be facilitated if guided by predictive and prognostic markers. As most cytotoxic drugs induce DNA damage, the DNA damage repair pathways hold potential for yielding such biomarkers. Here, we review the current evidence of a possible involvement of the nucleotide excision repair pathway in the efficacy of chemotherapeutic agents used in the treatment of colorectal cancer. Although a large number of studies have been conducted, they are generally of moderate size and heterogeneous in design. Up to date no firm conclusions can be drawn to translate these results into the clinic. We recommend further comprehensive investigations of the nucleotide excision repair pathway in large patient studies that include both discovery and validation cohorts. PMID:27183147
Excision repair characteristics of denV-transformed xeroderma pigmentosum cells.
Ley, R D; Applegate, L A; de Riel, J K; Henderson, E E
Introduction of the denV gene of phage T4, encoding the pyrimidine dimer-specific endonuclease V, into xeroderma pigmentosum cells XP12RO(M1) was reported to result in partial restoration of colony-forming ability and excision repair synthesis. We have further characterized 3 denV-transformed XP clones in terms of rates of excision of pyrimidine dimers and size of the resulting resynthesized regions following exposure to 100 J/m2 from an FS-40 sunlamp. In the denV-transformed XP cells we observed 50% dimer removal within 3-6 h after UV exposure as compared to no measurable removal in the XP12RO(M1) line and 50% dimer excision after 18 h in the GM637A human, control cells. Dimer removal was assayed with Micrococcus luteus UV-endonuclease in conjunction with sedimentation of treated DNA in alkaline sucrose gradients. The size of the resulting repaired regions was determined by the bromouracil photolysis technique. Based on the photolytic sensitivity of DNA repaired in the presence of bromodeoxyuridine, we calculated that the excision of a dimer in the GM637A cells appears to be accompanied by the resynthesis of a region approximately 95 nucleotides in length. Conversely, the resynthesized regions in the denV-transformed clones were considerably smaller and were estimated to be between 13 and 18 nucleotides in length. These results may indicate that either the endonuclease that initiated dimer repair dictated the size of the resynthesized region or that the long-patch repair observed in the normal cells resulted from the repair of non-dimer DNA lesions. PMID:2918865
Folate depletion impairs DNA excision repair in the colon of the rat
Choi, S; Kim, Y; Weitzel, J; Mason, J
Background/Aims—Diminished folate status appears to promote colonic carcinogenesis by, as of yet, undefined mechanisms. Impaired DNA repair plays a significant role in the evolution of many colon cancers. Since folate is essential for the de novo synthesis of nucleotides and since folate depletion has previously been associated with excessive DNA strand breaks, it was hypothesised that folate depletion may impair DNA repair. Studies were therefore performed to examine whether folate depletion affects the two major categories of DNA repair. Methods—Study 1: eight weanling male Sprague-Dawley rats were fed on diets containing either 0 or 8 mg folate/kg diet with 1% succinylsulphathiazole for four weeks. After viable colonocytes had been harvested, DNA excision repair was evaluated by a single cell gel electrophoresis assay. Study 2: eighteen animals were fed on similar diets for five weeks. Also in study 2, 18 additional rats were fed on the same defined diet without succinylsulphathiazole for 15 weeks. Weekly injections with the procarcinogen, 1,2-dimethylhydrazine (20 mg base/kg), were administered to the latter group of animals. Five microsatellite loci from different chromosomes were investigated for instability in hepatic and colonic DNA. Results—In study 1, a significantly retarded rate of DNA excision repair was observed in the folate deficient colonocytes compared with controls (p<0.05). In study 2, there was no evidence of instability at the five microsatellite loci associated with either short or long term folate depletion. Conclusions—Folate deficiency impairs DNA excision repair in rat colonic mucosa; a similar degree of deficiency, even when administered in conjunction with a colonic carcinogen, did not produce evidence of a widespread defect in mismatch repair. Keywords: folate; colon cancer; DNA repair; single cell gel electrophoresis; microsatellite instability; rat PMID:9771411
Fluorogenic DNA ligase and base excision repair enzyme assays using substrates labeled with single fluorophores.
Nikiforov, Theo T; Roman, Steven
Continuing our work on fluorogenic substrates labeled with single fluorophores for nucleic acid modifying enzymes, here we describe the development of such substrates for DNA ligases and some base excision repair enzymes. These substrates are hairpin-type synthetic DNA molecules with a single fluorophore located on a base close to the 3' ends, an arrangement that results in strong fluorescence quenching. When such substrates are subjected to an enzymatic reaction, the position of the dyes relative to that end of the molecules is altered, resulting in significant fluorescence intensity changes. The ligase substrates described here were 5' phosphorylated and either blunt-ended or carrying short, self-complementary single-stranded 5' extensions. The ligation reactions resulted in the covalent joining of the ends of the molecules, decreasing the quenching effect of the terminal bases on the dyes. To generate fluorogenic substrates for the base excision repair enzymes formamido-pyrimidine-DNA glycosylase (FPG), human 8-oxo-G DNA glycosylase/AP lyase (hOGG1), endonuclease IV (EndoIV), and apurinic/apyrimidinic endonuclease (APE1), we introduced abasic sites or a modified nucleotide, 8-oxo-dG, at such positions that their enzymatic excision would result in the release of a short fluorescent fragment. This was also accompanied by strong fluorescence increases. Overall fluorescence changes ranged from approximately 4-fold (ligase reactions) to more than 20-fold (base excision repair reactions). PMID:25728944
Global-genome Nucleotide Excision Repair Controlled by Ubiquitin/Sumo Modifiers
Rüthemann, Peter; Balbo Pogliano, Chiara; Naegeli, Hanspeter
Global-genome nucleotide excision repair (GG-NER) prevents genome instability by excising a wide range of different DNA base adducts and crosslinks induced by chemical carcinogens, ultraviolet (UV) light or intracellular side products of metabolism. As a versatile damage sensor, xeroderma pigmentosum group C (XPC) protein initiates this generic defense reaction by locating the damage and recruiting the subunits of a large lesion demarcation complex that, in turn, triggers the excision of aberrant DNA by endonucleases. In the very special case of a DNA repair response to UV radiation, the function of this XPC initiator is tightly controlled by the dual action of cullin-type CRL4DDB2 and sumo-targeted RNF111 ubiquitin ligases. This twofold protein ubiquitination system promotes GG-NER reactions by spatially and temporally regulating the interaction of XPC protein with damaged DNA across the nucleosome landscape of chromatin. In the absence of either CRL4DDB2 or RNF111, the DNA excision repair of UV lesions is inefficient, indicating that these two ubiquitin ligases play a critical role in mitigating the adverse biological effects of UV light in the exposed skin. PMID:27200078
Global-genome Nucleotide Excision Repair Controlled by Ubiquitin/Sumo Modifiers.
Rüthemann, Peter; Balbo Pogliano, Chiara; Naegeli, Hanspeter
Global-genome nucleotide excision repair (GG-NER) prevents genome instability by excising a wide range of different DNA base adducts and crosslinks induced by chemical carcinogens, ultraviolet (UV) light or intracellular side products of metabolism. As a versatile damage sensor, xeroderma pigmentosum group C (XPC) protein initiates this generic defense reaction by locating the damage and recruiting the subunits of a large lesion demarcation complex that, in turn, triggers the excision of aberrant DNA by endonucleases. In the very special case of a DNA repair response to UV radiation, the function of this XPC initiator is tightly controlled by the dual action of cullin-type CRL4(DDB2) and sumo-targeted RNF111 ubiquitin ligases. This twofold protein ubiquitination system promotes GG-NER reactions by spatially and temporally regulating the interaction of XPC protein with damaged DNA across the nucleosome landscape of chromatin. In the absence of either CRL4(DDB2) or RNF111, the DNA excision repair of UV lesions is inefficient, indicating that these two ubiquitin ligases play a critical role in mitigating the adverse biological effects of UV light in the exposed skin. PMID:27200078
FACT Assists Base Excision Repair by Boosting the Remodeling Activity of RSC
Ouararhni, Khalid; Roulland, Yohan; Ben Simon, Elsa; Kundu, Tapas; Hamiche, Ali; Angelov, Dimitar; Dimitrov, Stefan
FACT, in addition to its role in transcription, is likely implicated in both transcription-coupled nucleotide excision repair and DNA double strand break repair. Here, we present evidence that FACT could be directly involved in Base Excision Repair and elucidate the chromatin remodeling mechanisms of FACT during BER. We found that, upon oxidative stress, FACT is released from transcription related protein complexes to get associated with repair proteins and chromatin remodelers from the SWI/SNF family. We also showed the rapid recruitment of FACT to the site of damage, coincident with the glycosylase OGG1, upon the local generation of oxidized DNA. Interestingly, FACT facilitates uracil-DNA glycosylase in the removal of uracil from nucleosomal DNA thanks to an enhancement in the remodeling activity of RSC. This discloses a novel property of FACT wherein it has a co-remodeling activity and strongly enhances the remodeling capacity of the chromatin remodelers. Altogether, our data suggest that FACT may acts in concert with RSC to facilitate excision of DNA lesions during the initial step of BER. PMID:27467129
Nonspecific DNA Binding and Coordination of the First Two Steps of Base Excision Repair
Baldwin, Michael R.; O'Brien, Patrick J.
The base excision repair (BER) pathway repairs a wide variety of damaged nucleobases in DNA. This pathway is initiated by a DNA repair glycosylase, which locates the site of damage and catalyzes the excision of the damaged nucleobase. The resulting abasic site is further processed by apurinic/apyrimidinic site endonuclease 1 (APE1) to create a single strand nick with the 3'-hydroxyl that serves as a primer for DNA repair synthesis. Since an abasic site is highly mutagenic it is critical that the steps of the BER pathway be coordinated. Most human glycosylases bind tightly to their abasic product. APE1 displaces the bound glycosylase, thereby stimulating multiple turnover base excision. It has been proposed that direct protein-protein interactions are involved in the stimulation by APE1, but no common interaction motifs have been identified among the glycosylases that are stimulated by APE1. We characterized the APE1 stimulation of alkyladenine DNA glycosylase (AAG) using a variety of symmetric and asymmetric lesion-containing oligonucleotides. Efficient stimulation on a wide variety of substrates favors a model whereby both AAG and APE1 can simultaneously bind to DNA, but may not interact directly. Rather, nonspecific DNA binding by both AAG and APE1 enables APE1 to replace AAG at the abasic site. AAG is not displaced into solution, but remains bound to an adjacent undamaged site. We propose that nonspecific DNA binding interactions allow transient exposure of the abasic site so that it can be captured by APE1. PMID:20701268
Identification of a Second Locus in DROSOPHILA MELANOGASTER Required for Excision Repair
Boyd, J. B.; Snyder, R. D.; Harris, P. V.; Presley, J. M.; Boyd, S. F.; Smith, P. D.
The mus(2)201 locus in Drosophila is defined by two mutant alleles that render homozygous larvae hypersensitive to mutagens. Both alleles confer strong in vivo somatic sensitivity to treatment by methyl methanesulfonate, nitrogen mustard and ultraviolet radiation but only weak hypersensitivity to X-irradiation. Unlike the excision-defective mei-9 mutants identified in previous studies, the mus(2)201 mutants do not affect female fertility and do not appear to influence recombination proficiency or chromosome segregation in female meiocytes.—Three independent biochemical assays reveal that cell cultures derived from embryos homozygous for the mus(2)D1 allele are devoid of detectable excision repair. 1. Such cells quantitatively retain pyrimidine dimers in their DNA for 24 hr following UV exposure. 2. No measurable unscheduled DNA synthesis is induced in mutant cultures by UV treatment. 3. Single-strand DNA breaks, which are associated with normal excision repair after treatment with either UV or N-acetoxy-N-acetyl-2-aminofluorene,* are much reduced in these cultures. Mutant cells possess a normal capacity for postreplication repair and the repair of single-strand breaks induced by X-rays. PMID:6809529
FACT Assists Base Excision Repair by Boosting the Remodeling Activity of RSC.
Charles Richard, John Lalith; Shukla, Manu Shubhdarshan; Menoni, Hervé; Ouararhni, Khalid; Lone, Imtiaz Nisar; Roulland, Yohan; Papin, Christophe; Ben Simon, Elsa; Kundu, Tapas; Hamiche, Ali; Angelov, Dimitar; Dimitrov, Stefan
FACT, in addition to its role in transcription, is likely implicated in both transcription-coupled nucleotide excision repair and DNA double strand break repair. Here, we present evidence that FACT could be directly involved in Base Excision Repair and elucidate the chromatin remodeling mechanisms of FACT during BER. We found that, upon oxidative stress, FACT is released from transcription related protein complexes to get associated with repair proteins and chromatin remodelers from the SWI/SNF family. We also showed the rapid recruitment of FACT to the site of damage, coincident with the glycosylase OGG1, upon the local generation of oxidized DNA. Interestingly, FACT facilitates uracil-DNA glycosylase in the removal of uracil from nucleosomal DNA thanks to an enhancement in the remodeling activity of RSC. This discloses a novel property of FACT wherein it has a co-remodeling activity and strongly enhances the remodeling capacity of the chromatin remodelers. Altogether, our data suggest that FACT may acts in concert with RSC to facilitate excision of DNA lesions during the initial step of BER. PMID:27467129
Cellular homeostasis and repair in the mammalian liver.
Stanger, Ben Z
The mammalian liver is one of the most regenerative tissues in the body, capable of fully recovering mass and function after a variety of injuries. This factor alone makes the liver unusual among mammalian tissues, but even more atypical is the widely held notion that the method of repair depends on the manner of injury. Specifically, the liver is believed to regenerate via replication of existing cells under certain conditions and via differentiation from specialized cells--so-called facultative stem cells--under others. Nevertheless, despite the liver's dramatic and unique regenerative response, the cellular and molecular features of liver homeostasis and regeneration are only now starting to come into relief. This review provides an overview of normal liver function and development and focuses on the evidence for and against various models of liver homeostasis and regeneration. PMID:25668020
Gut Microbiota Imbalance and Base Excision Repair Dynamics in Colon Cancer
Ray, Debolina; Kidane, Dawit
Gut microbiota are required for host nutrition, energy balance, and regulating immune homeostasis, however, in some cases, this mutually beneficial relationship becomes twisted (dysbiosis), and the gut flora can incite pathological disorders including colon cancer. Microbial dysbiosis promotes the release of bacterial genotoxins, metabolites, and causes chronic inflammation, which promote oxidative DNA damage. Oxidized DNA base lesions are removed by base excision repair (BER), however, the role of this altered function of BER, as well as microbiota-mediated genomic instability and colon cancer development, is still poorly understood. In this review article, we will discuss how dysbiotic microbiota induce DNA damage, its impact on base excision repair capacity, the potential link of host BER gene polymorphism, and the risk of dysbiotic microbiota mediated genomic instability and colon cancer. PMID:27471558
Assays for chromatin remodeling during nucleotide excision repair in Saccharomyces cerevisiae
Zhang, Ling; Jones, Kristi; Smerdon, Michael J.; Gong, Feng
How DNA repair proteins interact with the dynamic structure of chromatin is an emerging question. Chromatin structure impedes the access of repair proteins to sites of DNA damage. Several recent studies have implicated chromatin remodeling complexes in DNA repair. In this report we summarize the methods we used to investigate chromatin remodeling during nucleotide excision repair (NER) in vivo. We describe a procedure to analyze UV-induced chromatin remodeling at the silent mating-type locus HML using isolated nuclei from UV treated yeast cells. In addition, a method to capture transient protein-protein associations in chromatin is outlined. We have used the methods described here to demonstrate that the SWI/SNF chromatin remodeling complex is involved in chromatin rearrangement during NER. PMID:19336254
Exploiting Base Excision Repair to Improve Therapeutic Approaches for Pancreatic Cancer
Sharbeen, George; McCarroll, Joshua; Goldstein, David; Phillips, Phoebe A.
Pancreatic ductal adenocarcinoma (PDA) is a highly chemoresistant and metastatic disease with a dismal 5-year survival rate of 6%. More effective therapeutic targets and approaches are urgently needed to tackle this devastating disease. The base excision repair (BER) pathway has been identified as a predictor of therapeutic response, prognostic factor, and therapeutic target in a variety of cancers. This review will discuss our current understanding of BER in PDA and its potential to improve PDA treatment. PMID:25988138
Removal of Misincorporated Ribonucleotides from Prokaryotic Genomes: An Unexpected Role for Nucleotide Excision Repair
Vaisman, Alexandra; McDonald, John P.; Huston, Donald; Kuban, Wojciech; Liu, Lili; Van Houten, Bennett; Woodgate, Roger
Stringent steric exclusion mechanisms limit the misincorporation of ribonucleotides by high-fidelity DNA polymerases into genomic DNA. In contrast, low-fidelity Escherichia coli DNA polymerase V (pol V) has relatively poor sugar discrimination and frequently misincorporates ribonucleotides. Substitution of a steric gate tyrosine residue with alanine (umuC_Y11A) reduces sugar selectivity further and allows pol V to readily misincorporate ribonucleotides as easily as deoxynucleotides, whilst leaving its poor base-substitution fidelity essentially unchanged. However, the mutability of cells expressing the steric gate pol V mutant is very low due to efficient repair mechanisms that are triggered by the misincorporated rNMPs. Comparison of the mutation frequency between strains expressing wild-type and mutant pol V therefore allows us to identify pathways specifically directed at ribonucleotide excision repair (RER). We previously demonstrated that rNMPs incorporated by umuC_Y11A are efficiently removed from DNA in a repair pathway initiated by RNase HII. Using the same approach, we show here that mismatch repair and base excision repair play minimal back-up roles in RER in vivo. In contrast, in the absence of functional RNase HII, umuC_Y11A-dependent mutagenesis increases significantly in ΔuvrA, uvrB5 and ΔuvrC strains, suggesting that rNMPs misincorporated into DNA are actively repaired by nucleotide excision repair (NER) in vivo. Participation of NER in RER was confirmed by reconstituting ribonucleotide-dependent NER in vitro. We show that UvrABC nuclease-catalyzed incisions are readily made on DNA templates containing one, two, or five rNMPs and that the reactions are stimulated by the presence of mispaired bases. Similar to NER of DNA lesions, excision of rNMPs proceeds through dual incisions made at the 8th phosphodiester bond 5′ and 4th–5th phosphodiester bonds 3′ of the ribonucleotide. Ribonucleotides misinserted into DNA can therefore be added to the
The Emerging Roles of ATP-Dependent Chromatin Remodeling Enzymes in Nucleotide Excision Repair
Czaja, Wioletta; Mao, Peng; Smerdon, Michael J.
DNA repair in eukaryotic cells takes place in the context of chromatin, where DNA, including damaged DNA, is tightly packed into nucleosomes and higher order chromatin structures. Chromatin intrinsically restricts accessibility of DNA repair proteins to the damaged DNA and impacts upon the overall rate of DNA repair. Chromatin is highly responsive to DNA damage and undergoes specific remodeling to facilitate DNA repair. How damaged DNA is accessed, repaired and restored to the original chromatin state, and how chromatin remodeling coordinates these processes in vivo, remains largely unknown. ATP-dependent chromatin remodelers (ACRs) are the master regulators of chromatin structure and dynamics. Conserved from yeast to humans, ACRs utilize the energy of ATP to reorganize packing of chromatin and control DNA accessibility by sliding, ejecting or restructuring nucleosomes. Several studies have demonstrated that ATP-dependent remodeling activity of ACRs plays important roles in coordination of spatio-temporal steps of different DNA repair pathways in chromatin. This review focuses on the role of ACRs in regulation of various aspects of nucleotide excision repair (NER) in the context of chromatin. We discuss current understanding of ATP-dependent chromatin remodeling by various subfamilies of remodelers and regulation of the NER pathway in vivo. PMID:23109894
3CAPS – a structural AP–site analogue as a tool to investigate DNA base excision repair
Schuermann, David; Scheidegger, Simon P.; Weber, Alain R.; Bjørås, Magnar; Leumann, Christian J.; Schär, Primo
Abasic sites (AP-sites) are frequent DNA lesions, arising by spontaneous base hydrolysis or as intermediates of base excision repair (BER). The hemiacetal at the anomeric centre renders them chemically reactive, which presents a challenge to biochemical and structural investigation. Chemically more stable AP-site analogues have been used to avoid spontaneous decay, but these do not fully recapitulate the features of natural AP–sites. With its 3′–phosphate replaced by methylene, the abasic site analogue 3CAPS was suggested to circumvent some of these limitations. Here, we evaluated the properties of 3CAPS in biochemical BER assays with mammalian proteins. 3CAPS-containing DNA substrates were processed by APE1, albeit with comparably poor efficiency. APE1-cleaved 3CAPS can be extended by DNA polymerase β but repaired only by strand displacement as the 5′–deoxyribophosphate (dRP) cannot be removed. DNA glycosylases physically and functionally interact with 3CAPS substrates, underlining its structural integrity and biochemical reactivity. The AP lyase activity of bifunctional DNA glycosylases (NTH1, NEIL1, FPG), however, was fully inhibited. Notably, 3CAPS-containing DNA also effectively inhibited the activity of bifunctional glycosylases on authentic substrates. Hence, the chemically stable 3CAPS with its preserved hemiacetal functionality is a potent tool for BER research and a potential inhibitor of bifunctional DNA glycosylases. PMID:26733580
Impact of ribonucleotide incorporation by DNA polymerases β and λ on oxidative base excision repair
Crespan, Emmanuele; Furrer, Antonia; Rösinger, Marcel; Bertoletti, Federica; Mentegari, Elisa; Chiapparini, Giulia; Imhof, Ralph; Ziegler, Nathalie; Sturla, Shana J.; Hübscher, Ulrich; van Loon, Barbara; Maga, Giovanni
Oxidative stress is a very frequent source of DNA damage. Many cellular DNA polymerases (Pols) can incorporate ribonucleotides (rNMPs) during DNA synthesis. However, whether oxidative stress-triggered DNA repair synthesis contributes to genomic rNMPs incorporation is so far not fully understood. Human specialized Pols β and λ are the important enzymes involved in the oxidative stress tolerance, acting both in base excision repair and in translesion synthesis past the very frequent oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxo-G). We found that Pol β, to a greater extent than Pol λ can incorporate rNMPs opposite normal bases or 8-oxo-G, and with a different fidelity. Further, the incorporation of rNMPs opposite 8-oxo-G delays repair by DNA glycosylases. Studies in Pol β- and λ-deficient cell extracts suggest that Pol β levels can greatly affect rNMP incorporation opposite oxidative DNA lesions. PMID:26917111
Structural and Functional Studies on Nucleotide Excision Repair From Recognition to Incision.
Caroline Kisker
Maintenance of the correct genetic information is crucial for all living organisms because mutations are the primary cause of hereditary diseases, as well as cancer and may also be involved in aging. The importance of genomic integrity is underscored by the fact that 80 to 90% of all human cancers are ultimately due to DNA damage. Among the different repair mechanisms that have evolved to protect the genome, nucleotide excision repair (NER) is a universal pathway found in all organisms. NER removes a wide variety of bulky DNA adducts including the carcinogenic cyclobutane pyrimidine dimers induced by UV radiation, benzo(a)pyrene-guanine adducts caused by smoking and the guanine-cisplatin adducts induced by chemotherapy. The importance of this repair mechanism is reflected by three severe inherited diseases in humans, which are due to defects in NER: xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy.
Base Excision Repair in Physiology and Pathology of the Central Nervous System
Bosshard, Matthias; Markkanen, Enni; van Loon, Barbara
Relatively low levels of antioxidant enzymes and high oxygen metabolism result in formation of numerous oxidized DNA lesions in the tissues of the central nervous system. Accumulation of damage in the DNA, due to continuous genotoxic stress, has been linked to both aging and the development of various neurodegenerative disorders. Different DNA repair pathways have evolved to successfully act on damaged DNA and prevent genomic instability. The predominant and essential DNA repair pathway for the removal of small DNA base lesions is base excision repair (BER). In this review we will discuss the current knowledge on the involvement of BER proteins in the maintenance of genetic stability in different brain regions and how changes in the levels of these proteins contribute to aging and the onset of neurodegenerative disorders. PMID:23203191
Cloning, comparative mapping, and RNA expression of the mouse homologues of the Saccharomyces cerevisiae nucleotide excision repair gene RAD23.
van der Spek, P J; Visser, C E; Hanaoka, F; Smit, B; Hagemeijer, A; Bootsma, D; Hoeijmakers, J H
The Saccharomyces cerevisiae RAD23 gene is involved in nucleotide excision repair (NER). Two human homologs of RAD23, HHR23A and HHR23B (HGMW-approved symbols RAD23A and RAD23B), were previously isolated. The HHR23B protein is complexed with the protein defective in the cancer-prone repair syndrome xeroderma pigmentosum, complementation group C, and is specifically involved in the global genome NER subpathway. The cloning of both mouse homologs (designated MHR23A and MHR23B) and detailed sequence comparison permitted the deduction of the following overall structure for all RAD23 homologs: an ubiquitin-like N-terminus followed by a strongly conserved 50-amino-acid domain that is repeated at the C-terminus. We also found this domain as a specific C-terminal extension of one of the ubiquitin-conjugating enzymes, providing a second link with the ubiquitin pathway. By means of in situ hybridization, MHR23A was assigned to mouse chromosome 8C3 and MHR23B to 4B3. Because of the close chromosomal proximity of human XPC and HHR23B, the mouse XPC chromosomal location was determined (6D). Physical disconnection of the genes in mouse argues against a functional significance of the colocalization of these genes in human. Northern blot analysis revealed constitutive expression of both MHR23 genes in all tissues examined. Elevated RNA expression of both MHR23 genes was observed in testis. Although the RAD23 equivalents are well conserved during evolution, the mammalian genes did not express the UV-inducible phenotype of their yeast counterpart. This may point to a fundamental difference between the UV responses of yeast and human. No stage-specific mRNA expression during the cell cycle was observed for the mammalian RAD23 homologs. PMID:8808275
DREMECELS: A Curated Database for Base Excision and Mismatch Repair Mechanisms Associated Human Malignancies
Shukla, Ankita; Singh, Tiratha Raj
DNA repair mechanisms act as a warrior combating various damaging processes that ensue critical malignancies. DREMECELS was designed considering the malignancies with frequent alterations in DNA repair pathways, that is, colorectal and endometrial cancers, associated with Lynch syndrome (also known as HNPCC). Since lynch syndrome carries high risk (~40–60%) for both cancers, therefore we decided to cover all three diseases in this portal. Although a large population is presently affected by these malignancies, many resources are available for various cancer types but no database archives information on the genes specifically for only these cancers and disorders. The database contains 156 genes and two repair mechanisms, base excision repair (BER) and mismatch repair (MMR). Other parameters include some of the regulatory processes that have roles in these disease progressions due to incompetent repair mechanisms, specifically BER and MMR. However, our unique database mainly provides qualitative and quantitative information on these cancer types along with methylation, drug sensitivity, miRNAs, copy number variation (CNV) and somatic mutations data. This database would serve the scientific community by providing integrated information on these disease types, thus sustaining diagnostic and therapeutic processes. This repository would serve as an excellent accompaniment for researchers and biomedical professionals and facilitate in understanding such critical diseases. DREMECELS is publicly available at http://www.bioinfoindia.org/dremecels. PMID:27276067
Unscheduled DNA Synthesis: The Clinical and Functional Assay for Global Genomic DNA Nucleotide Excision Repair
Latimer, Jean J.; Kelly, Crystal M.
The unscheduled DNA synthesis (UDS) assay measures the ability of a cell to perform global genomic nucleotide excision repair (NER). This chapter provides instructions for the application of this technique by creating 6-4 photoproducts and pyrimidine dimers using UV-C irradiation. This procedure is designed specifically for quantification of the 6-4 photoproducts. Repair is quantified by the amount of radioactive thymidine incorporated during repair synthesis after this insult, and radioactivity is evaluated by grain counting after autoradiography. The results are used to clinically diagnose human DNA repair deficiency disorders and provide a basis for investigation of repair deficiency in human tissues or tumors. No other functional assay is available that directly measures the capacity to perform NER on the entire genome without the use of specific antibodies. Since live cells are required for this assay, explant culture techniques must be previously established. Host cell reactivation (HCR), as discussed in Chapter 37, is not an equivalent technique, as it measures only transcription-coupled repair (TCR) at active genes, a small subset of total NER. PMID:24623250
Repair of traumatized mammalian hair cells via sea anemone repair proteins.
Tang, Pei-Ciao; Smith, Karen Müller; Watson, Glen M
Mammalian hair cells possess only a limited ability to repair damage after trauma. In contrast, sea anemones show a marked capability to repair damaged hair bundles by means of secreted repair proteins (RPs). Previously, it was found that recovery of traumatized hair cells in blind cavefish was enhanced by anemone-derived RPs; therefore, the ability of anemone RPs to assist recovery of damaged hair cells in mammals was tested here. After a 1 h incubation in RP-enriched culture media, uptake of FM1-43 by experimentally traumatized murine cochlear hair cells was restored to levels comparable to those exhibited by healthy controls. In addition, RP-treated explants had significantly more normally structured hair bundles than time-matched traumatized control explants. Collectively, these results indicate that anemone-derived RPs assist in restoring normal function and structure of experimentally traumatized hair cells of the mouse cochlea. PMID:27489215
Transcription-coupled nucleotide excision repair factors promote R-loop-induced genome instability
Sollier, Julie; Stork, Caroline Townsend; García-Rubio, María L.; Paulsen, Renee D.; Aguilera, Andrés; Cimprich, Karlene A.
Summary R-loops, consisting of an RNA-DNA hybrid and displaced single-stranded DNA, are physiological structures that regulate various cellular processes occurring on chromatin. Intriguingly, changes in R-loop dynamics have also been associated with DNA damage accumulation and genome instability, however the mechanisms underlying R-loop induced DNA damage remain unknown. Here we demonstrate in human cells that R-loops induced by the absence of diverse RNA processing factors, including the RNA/DNA helicases Aquarius (AQR) and Senataxin (SETX), or by the inhibition of topoisomerase I, are actively processed into DNA double-strand breaks (DSBs) by the nucleotide excision repair endonucleases XPF and XPG. Surprisingly, DSB formation requires the transcription-coupled nucleotide excision repair (TC-NER) factor Cockayne syndrome group B (CSB), but not the global genome repair protein XPC. These findings reveal an unexpected and potentially deleterious role for TC-NER factors in driving R-loop-induced DNA damage and genome instability. PMID:25435140
Evidence for indirect involvement of thymidine kinase in excision repair processes in mouse cell lines
McKenna, P.G.; Yasseen, A.A.; McKelvey, V.J.
Wild-type cells and thymidine kinase-deficient clones from two mouse lymphoma cell lines, P388 and L5178Y, were compared for sensitivity to killing by the mutagens, ultraviolet irradiation (UV), ethyl methane sulfonate (EMS), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Two out of three thymidine kinase-deficient P388 clones showed significantly enhanced sensitivity to killing by all three mutagens. This increased sensitivity to killing was also reflected in increased mutagenesis by the three mutagens. In the L5178Y cell line, wild-type cells showed little difference to two thymidine kinase-deficient clones in terms of mutagen sensitivity. This indicates that thymidine kinase may be significant for DNA repair processes in P388 but not in L5178Y cells. Unscheduled DNA synthesis (UDS) experiments were carried out on P388 and L5178Y wild-type cells and wild-type Friend leukemia cells (which are mutagen-sensitive when deficient in thymidine kinase). The UDS experiments showed the L5178Y cells were low in excision repair abilities relative to the P388 cells and the Friend cell clone. This indicates that the increased mutagen sensitivity in thymidine kinase-deficient P388 and clone 707 Friend cells may be due to thymidine kinase playing an indirect role in DNA excision repair, a process which is of little significance in the L5178Y cell line.
Nucleotide excision repair is impaired by binding of transcription factors to DNA.
Sabarinathan, Radhakrishnan; Mularoni, Loris; Deu-Pons, Jordi; Gonzalez-Perez, Abel; López-Bigas, Núria
Somatic mutations are the driving force of cancer genome evolution. The rate of somatic mutations appears to be greatly variable across the genome due to variations in chromatin organization, DNA accessibility and replication timing. However, other variables that may influence the mutation rate locally are unknown, such as a role for DNA-binding proteins, for example. Here we demonstrate that the rate of somatic mutations in melanomas is highly increased at active transcription factor binding sites and nucleosome embedded DNA, compared to their flanking regions. Using recently available excision-repair sequencing (XR-seq) data, we show that the higher mutation rate at these sites is caused by a decrease of the levels of nucleotide excision repair (NER) activity. Our work demonstrates that DNA-bound proteins interfere with the NER machinery, which results in an increased rate of DNA mutations at the protein binding sites. This finding has important implications for our understanding of mutational and DNA repair processes and in the identification of cancer driver mutations. PMID:27075101
ALKBH1 is dispensable for abasic site cleavage during base excision repair and class switch recombination.
Müller, Tina A; Yu, Kefei; Hausinger, Robert P; Meek, Katheryn
Potential roles of the abasic site lyase activity associated with AlkB homolog 1 (ALKBH1) were assessed by studies focusing on the two cellular processes that create abasic sites as intermediates: base excision repair and class switch recombination. Alkbh1(-/-) pups (lacking exon 3) were born at a lower than expected frequency from heterozygous parents, suggesting a reduced survival rate and non-Mendelian inheritance, and they exhibited a gender bias in favor of males (70% males and 30% females). To study ALKBH1's potential involvement in DNA repair, fibroblasts were isolated from Alkbh1(-/-) mice, spontaneously immortalized and tested for resistance to DNA damaging agents. Alkbh1(-/-) and isogenic cells expressing hALKBH1 showed no difference in survival to the DNA damaging agents methyl-methionine sulfate or H2O2. This result indicates that ALKBH1 does not play a major role in the base excision repair pathway. To assess ALKBH1's role in class switch recombination, splenic B cells were isolated from Alkbh1(-/-) and Alkbh1(+/+) mice and subjected to switching from IgM to IgG1. No differences were found in IgG1 switching, suggesting that Alkbh1 is not involved in class switch recombination of the immunoglobulin heavy chain during B lymphocyte activation. PMID:23825659
New design of nucleotide excision repair (NER) inhibitors for combination cancer therapy.
Gentile, Francesco; Tuszynski, Jack A; Barakat, Khaled H
Many cancer chemotherapy agents act by targeting the DNA of cancer cells, causing substantial damage within their genome and causing them to undergo apoptosis. An effective DNA repair pathway in cancer cells can act in a reverse way by removing these drug-induced DNA lesions, allowing cancer cells to survive, grow and proliferate. In this context, DNA repair inhibitors opened a new avenue in cancer treatment, by blocking the DNA repair mechanisms from removing the chemotherapy-mediated DNA damage. In particular, the nucleotide excision repair (NER) involves more than thirty protein-protein interactions and removes DNA adducts caused by platinum-based chemotherapy. The excision repair cross-complementation group 1 (ERCC1)-xeroderma pigmentosum, complementation group A (XPA) protein (XPA-ERCC1) complex seems to be one of the most promising targets in this pathway. ERCC1 is over expressed in cancer cells and the only known cellular function so far for XPA is to recruit ERCC1 to the damaged point. Here, we build upon our recent advances in identifying inhibitors for this interaction and continue our efforts to rationally design more effective and potent regulators for the NER pathway. We employed in silico drug design techniques to: (1) identify compounds similar to the recently discovered inhibitors, but more effective at inhibiting the XPA-ERCC1 interactions, and (2) identify different scaffolds to develop novel lead compounds. Two known inhibitor structures have been used as starting points for two ligand/structure-hybrid virtual screening approaches. The findings described here form a milestone in discovering novel inhibitors for the NER pathway aiming at improving the efficacy of current platinum-based therapy, by modulating the XPA-ERCC1 interaction. PMID:26939044
Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair.
Han, Chunhua; Wani, Gulzar; Zhao, Ran; Qian, Jiang; Sharma, Nidhi; He, Jinshan; Zhu, Qianzheng; Wang, Qi-En; Wani, Altaf A
Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair endonuclease, which cleaves DNA strands on the 3' side of the DNA damage during nucleotide excision repair (NER). XPG also plays a crucial role in initiating DNA repair synthesis through recruitment of PCNA to the repair sites. However, the fate of XPG protein subsequent to the excision of DNA damage has remained unresolved. Here, we show that XPG, following its action on bulky lesions resulting from exposures to UV irradiation and cisplatin, is subjected to proteasome-mediated proteolytic degradation. Productive NER processing is required for XPG degradation as both UV and cisplatin treatment-induced XPG degradation is compromised in NER-deficient XP-A, XP-B, XP-C, and XP-F cells. In addition, the NER-related XPG degradation requires Cdt2, a component of an E3 ubiquitin ligase, CRL4(Cdt2). Micropore local UV irradiation and in situ Proximity Ligation assays demonstrated that Cdt2 is recruited to the UV-damage sites and interacts with XPG in the presence of PCNA. Importantly, Cdt2-mediated XPG degradation is crucial to the subsequent recruitment of DNA polymerase δ and DNA repair synthesis. Collectively, our data support the idea of PCNA recruitment to damage sites which occurs in conjunction with XPG, recognition of the PCNA-bound XPG by CRL4(Cdt2) for specific ubiquitylation and finally the protein degradation. In essence, XPG elimination from DNA damage sites clears the chromatin space needed for the subsequent recruitment of DNA polymerase δ to the damage site and completion of gap-filling DNA synthesis during the final stage of NER. PMID:25483071
Mutations in XPA that prevent association with ERCC1 are defective in nucleotide excision repair.
Li, L; Peterson, C A; Lu, X; Legerski, R J
The human repair proteins XPA and ERCC1 have been shown to be absolutely required for the incision step of nucleotide excision repair, and recently we identified an interaction between these two proteins both in vivo and in vitro (L. Li, S. J. Elledge, C. A. Peterson, E. S. Bales, and R. J. Legerski, Proc. Natl. Acad. Sci. USA 91:5012-5016, 1994). In this report, we demonstrate the functional relevance of this interaction. The ERCC1-binding domain on XPA was previously mapped to a region containing two highly conserved XPA sequences, Gly-72 to Phe-75 and Glu-78 to Glu-84, which are termed the G and E motifs, respectively. Site-specific mutagenesis was used to independently delete these motifs and create two XPA mutants referred to as delta G and delta E. In vitro, the binding of ERCC1 to delta E was reduced by approximately 70%, and binding to delta G was undetectable; furthermore, both mutants failed to complement XPA cell extracts in an in vitro DNA repair synthesis assay. In vivo, the delta E mutant exhibited an intermediate level of complementation of XPA cells and the delta G mutant exhibited little or no complementation. In addition, the delta G mutant inhibited repair synthesis in wild-type cell extracts, indicating that it is a dominant negative mutant. The delta E and delta G mutations, however, did not affect preferential binding of XPA to damaged DNA. These results suggest that the association between XPA and ERCC1 is a required step in the nucleotide excision repair pathway and that the probable role of the interaction is to recruit the ERCC1 incision complex to the damage site. Finally, the affinity of the XPA-ERCC1 complex was found to increase as a function of salt concentration, indicating a hydrophobic interaction; the half-life of the complex was determined to be approximately 90 min. PMID:7891694
A Case of Microangiopathic Hemolytic Anemia after Myxoma Excision and Mitral Valve Repair Presenting as Hemolytic Uremic Syndrome
Park, Young Joo; Kim, Sang Pil; Shin, Ho-Jin
Microangiopathic hemolytic anemia occurs in a diverse group of disorders, including thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, and prosthetic cardiac valves. Hemolytic anemia also occurs as a rare complication after mitral valve repair. In this report, we describe a case of microangiopathic hemolytic anemia following myxoma excision and mitral valve repair, which was presented as hemolytic uremic syndrome. PMID:27081450
A Case of Microangiopathic Hemolytic Anemia after Myxoma Excision and Mitral Valve Repair Presenting as Hemolytic Uremic Syndrome.
Park, Young Joo; Kim, Sang Pil; Shin, Ho-Jin; Choi, Jung Hyun
Microangiopathic hemolytic anemia occurs in a diverse group of disorders, including thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, and prosthetic cardiac valves. Hemolytic anemia also occurs as a rare complication after mitral valve repair. In this report, we describe a case of microangiopathic hemolytic anemia following myxoma excision and mitral valve repair, which was presented as hemolytic uremic syndrome. PMID:27081450
Nucleotide excision repair and the 26S proteasome function together to promote trinucleotide repeat expansions.
Concannon, Claire; Lahue, Robert S
Trinucleotide repeat (TNR) expansion underpins a number of inheritable neurological human disorders. Multiple mechanisms are thought to contribute to the expansion process. The incorrect processing of the repeat tract by DNA repair proteins can drive this mutation process forward, as expansions are suppressed following ablation of certain repair factors in mouse models and cell models of disease. Nucleotide excision repair (NER) is one repair pathway implicated in TNR instability, although most previous work focussed on TNR contractions, not expansions. Here we investigated the role of NER in modulating expansions of threshold-length (CTG·CAG) repeats in yeast. We show that both the global genome and transcription-coupled repair subpathways promote expansions of threshold-length TNRs. Furthermore, NER works with the 26S proteasome to drive expansions, based on analysis of double mutants defective in both pathways, and of Rad23, a protein involved in both NER and the shuttling of ubiquitinated proteins to the proteasome. This work provides the first evidence that both subpathways of NER can promote threshold-length TNR expansions and that NER interacts with the proteasome to drive expansions. PMID:24359926
UV-induced DNA excision repair in rat fibroblasts during immortalization and terminal differentiation in vitro
Vijg, J.; Mullaart, E.; Berends, F.; Lohman, P.H.; Knook, D.L.
UV-induced DNA excision repair was studied as DNA repair synthesis and dimer removal in rat fibroblast cultures, initiated from either dense or sparse inocula of primary cells grown from skin biopsies. During passaging in vitro an initial increase in DNA repair synthesis, determined both autoradiographically as unscheduled DNA synthesis (UDS) and by means of the BrdU photolysis assay as the number and average size of repair patches, was found to be associated with a morphological shift from small spindle-shaped to large pleiomorphic cells observed over the first twenty generations. In cell populations in growth crisis, a situation exclusively associated with thin-inoculum cultures in which the population predominantly consisted of large pleiomorphic cells, UDS was found to occur at a low level. After development of secondary cultures into immortal cell lines, both repair synthesis and morphology appeared to be the same as in the original primary spindle-shaped cells. At all passages the capacity to remove UV-induced pyrimidine dimers was found to be low, as indicated by the persistence of Micrococcus luteus UV endonuclease-sensitive sites. These results are discussed in the context of terminal differentiation and immortalization of rat fibroblasts upon establishment in vitro.
MCM9 Is Required for Mammalian DNA Mismatch Repair.
Traver, Sabine; Coulombe, Philippe; Peiffer, Isabelle; Hutchins, James R A; Kitzmann, Magali; Latreille, Daniel; Méchali, Marcel
DNA mismatch repair (MMR) is an evolutionarily conserved process that corrects DNA polymerase errors during replication to maintain genomic integrity. In E. coli, the DNA helicase UvrD is implicated in MMR, yet an analogous helicase activity has not been identified in eukaryotes. Here, we show that mammalian MCM9, a protein involved in replication and homologous recombination, forms a complex with MMR initiation proteins (MSH2, MSH3, MLH1, PMS1, and the clamp loader RFC) and is essential for MMR. Mcm9-/- cells display microsatellite instability and MMR deficiency. The MCM9 complex has a helicase activity that is required for efficient MMR since wild-type but not helicase-dead MCM9 restores MMR activity in Mcm9-/- cells. Moreover, MCM9 loading onto chromatin is MSH2-dependent, and in turn MCM9 stimulates the recruitment of MLH1 to chromatin. Our results reveal a role for MCM9 and its helicase activity in mammalian MMR. PMID:26300262
Investigation of bacterial nucleotide excision repair using single-molecule techniques.
Van Houten, Bennett; Kad, Neil
Despite three decades of biochemical and structural analysis of the prokaryotic nucleotide excision repair (NER) system, many intriguing questions remain with regard to how the UvrA, UvrB, and UvrC proteins detect, verify and remove a wide range of DNA lesions. Single-molecule techniques have begun to allow more detailed understanding of the kinetics and action mechanism of this complex process. This article reviews how atomic force microscopy and fluorescence microscopy have captured new glimpses of how these proteins work together to mediate NER. PMID:24472181
Relationship of DNA repair processes to mutagenesis and carcinogenesis in mammalian cells. Progress report, August 1, 1977-October 31, 1980
Evans, H.H.
The objective of this research is to determine the role of DNA repair in mutagenesis and carcinogenesis in mammalian cells. More specifically, mutant strains will be selected which are deficient in various DNA repair pathways. These strains will be studied with regard to (1) the nature of the defect in repair, and (2) the mutability and transformability of the defective cells by various agents as compared to the wild type parental cells. The results to date include progress in the following areas: (1) determination of optimum conditions for growth and maintenance of cells and for quantitative measurement of various cellular parameters; (2) investigation of the effect of holding mutagenized cells for various periods in a density inhibited state on survival and on mutation and transformation frequencies; (3) examination of the repair capabilities of BHK cells, as compared to repair-proficient and repair-deficient human cells and excision-deficient mouse cells, as measured by the reactivation of Herpes simplex virus (HSV) treated with radiation and ethylmethane sulfonate (EMS); (4) initiation of host cell reactivation viral sucide enrichment and screening of survivors of the enrichment for sensitivity to ionizing radiation; and (5) investigation of the toxicity, mutagenicity, and carcinogenicity of various metabolites of 4-nitroquinoline-1-oxide (4-NQO). (ERB)
E2F1 and p53 Transcription Factors as Accessory Factors for Nucleotide Excision Repair
Vélez-Cruz, Renier; Johnson, David G.
Many of the biochemical details of nucleotide excision repair (NER) have been established using purified proteins and DNA substrates. In cells however, DNA is tightly packaged around histones and other chromatin-associated proteins, which can be an obstacle to efficient repair. Several cooperating mechanisms enhance the efficiency of NER by altering chromatin structure. Interestingly, many of the players involved in modifying chromatin at sites of DNA damage were originally identified as regulators of transcription. These include ATP-dependent chromatin remodelers, histone modifying enzymes and several transcription factors. The p53 and E2F1 transcription factors are well known for their abilities to regulate gene expression in response to DNA damage. This review will highlight the underappreciated, transcription-independent functions of p53 and E2F1 in modifying chromatin structure in response to DNA damage to promote global NER. PMID:23202967
Vertebrate POLQ and POLβ Cooperate in Base Excision Repair of Oxidative DNA Damage
Yoshimura, Michio; Kohzaki, Masaoki; Nakamura, Jun; Asagoshi, Kenjiro; Sonoda, Eiichiro; Hou, Esther; Prasad, Rajendra; Wilson, Samuel H.; Tano, Keizo; Yasui, Akira; Lan, Li; Seki, Mineaki; Wood, Richard D.; Arakawa, Hiroshi; Buerstedde, Jean-Marie; Hochegger, Helfrid; Okada, Takashi; Hiraoka, Masahiro; Takeda, Shunichi
Summary Base excision repair (BER) plays an essential role in protecting cells from mutagenic base damage caused by oxidative stress, hydrolysis, and environmental factors. POLQ is a DNA polymerase, which appears to be involved in translesion DNA synthesis (TLS) past base damage. We disrupted POLQ, and its homologs HEL308 and POLN in chicken DT40 cells, and also created polq/hel308 and polq/poln double mutants. We found that POLQ-deficient mutants exhibit hypersensitivity to oxidative base damage induced by H2O2, but not to UV or cisplatin. Surprisingly, this phenotype was synergistically increased by concomitant deletion of the major BER polymerase, POLβ. Moreover, extracts from a polq null mutant cell line show reduced BER activity, and POLQ, like POLβ, accumulated rapidly at sites of base damage. Accordingly, POLQ and POLβ share an overlapping function in the repair of oxidative base damage. Taken together, these results suggest a role for vertebrate POLQ in BER. PMID:17018297
SUMO and ubiquitin-dependent XPC exchange drives nucleotide excision repair
van Cuijk, Loes; van Belle, Gijsbert J.; Turkyilmaz, Yasemin; Poulsen, Sara L.; Janssens, Roel C.; Theil, Arjan F.; Sabatella, Mariangela; Lans, Hannes; Mailand, Niels; Houtsmuller, Adriaan B.; Vermeulen, Wim; Marteijn, Jurgen A.
XPC recognizes UV-induced DNA lesions and initiates their removal by nucleotide excision repair (NER). Damage recognition in NER is tightly controlled by ubiquitin and SUMO modifications. Recent studies have shown that the SUMO-targeted ubiquitin ligase RNF111 promotes K63-linked ubiquitylation of SUMOylated XPC after DNA damage. However, the exact regulatory function of these modifications in vivo remains elusive. Here we show that RNF111 is required for efficient repair of ultraviolet-induced DNA lesions. RNF111-mediated ubiquitylation promotes the release of XPC from damaged DNA after NER initiation, and is needed for stable incorporation of the NER endonucleases XPG and ERCC1/XPF. Our data suggest that RNF111, together with the CRL4DDB2 ubiquitin ligase complex, is responsible for sequential XPC ubiquitylation, which regulates the recruitment and release of XPC and is crucial for efficient progression of the NER reaction, thereby providing an extra layer of quality control of NER. PMID:26151477
A physical interaction of UvrD with nucleotide excision repair protein UvrB.
Ahn, B
The dual-incision nature of the reaction of UV-irradiated DNA catalyzed by the UvrABC complex potentially leads to excision of a damaged fragment. However, neither fragment release under nondenaturing conditions nor the UvrBC proteins are turned over. The addition of the UvrD protein to the incised DNA-UvrBC complex results in excision of the incised damaged strand and in the turnover of the UvrC protein. In an effort to better understand the involvement of UvrD in the excision step, immunoprecipitation was used to detect interacting proteins with UvrD in the DNA repair. In this communication, it is shown that UvrA and UvrB are precipitated with UvrD in solution but the UvrAB complex is not. In the incision complex, UvrB could be precipitated and the preincubation of UvrD with UvrB revealed an inhibitory effect on the turnover of the incision complex. These data imply that UvrB in the incision complex seems to recruit UvrD to the 3' incised site of the incised strand by protein-protein interaction and to allow initiation of unwinding by UvrD from the resulting nick in a 3' to 5' direction. PMID:11101153
Excision repair of 5,6-dihydroxydihydrothymine from the DNA of Micrococcus radiodurans
Targovnik, H.S.; Hariharan, P.V.
One of the major ionizing radiation products, 5,6-dihydroxydihydrothymine (thymine glycol), was measured in the DNA of Micrococcus radiodurans following exposure of cells to 6.8-MeV electrons or 254-nm ultraviolet light. Removal of 5,6-dihydroxydihydrothymine was measured in both an ionizing radiation-sensitive strain (262) and a highly radioresistant strain (the wild type W/sup +/) of Micrococcus radiodurans. Within 30 min of incubation (33/sup 0/C) following exposure to ultraviolet light (2400 J/m/sup 2/) approximately 60% of the thymine glycols were excised, whereas in the case of ionizing radiation (250 krad) only 35% were removed from the cellular DNA of the wild-type strain. In contrast less than 50% of the thymine glycols were excised from the sensitive strain. The amount of DNA degradation induced by radiation was less than 10% in both strains. The results suggest a possible correlation between reduced excision repair of base damage and increased radiation sensitivity.
Polymorphisms within base and nucleotide excision repair pathways and risk of differentiated thyroid carcinoma.
Cipollini, Monica; Figlioli, Gisella; Maccari, Giuseppe; Garritano, Sonia; De Santi, Chiara; Melaiu, Ombretta; Barone, Elisa; Bambi, Franco; Ermini, Stefano; Pellegrini, Giovanni; Cristaudo, Alfonso; Foddis, Rudy; Bonotti, Alessandra; Romei, Cristina; Vivaldi, Agnese; Agate, Laura; Molinari, Eleonora; Barale, Roberto; Forsti, Asta; Hemminki, Kari; Elisei, Rossella; Gemignani, Federica; Landi, Stefano
The thyrocytes are exposed to high levels of oxidative stress which could induce DNA damages. Base excision repair (BER) is one of the principal mechanisms of defense against oxidative DNA damage, however recent evidences suggest that also nucleotide excision repair (NER) could be involved. The aim of present work was to identify novel differentiated thyroid cancer (DTC) risk variants in BER and NER genes. For this purpose, the most strongly associated SNPs within NER and BER genes found in our previous GWAS on DTC were selected and replicated in an independent series of samples for a new case-control study. Although a positive signal was detected at the nominal level of 0.05 for rs7689099 (encoding for an aminoacid change proline to arginine at codon 117 within NEIL3), none of the considered SNPs (i.e. rs7990340 and rs690860 within RFC3, rs3744767 and rs1131636 within RPA1, rs16962916 and rs3136166 in ERCC4, and rs17739370 and rs7689099 in NEIL3) was associated with the risk of DTC when the correction of multiple testing was applied. In conclusion, a role of NER and BER pathways was evoked in the susceptibility to DTC. However, this seemed to be limited to few polymorphic genes and the overall effect size appeared weak. PMID:27062014
Molecular cloning of the human nucleotide-excision-repair gene ERCC4
Thompson, L.H.; Brookman, K.W.; Weber, C.A.; Salazar, E.P.; Reardon, J.T.; Sancar, A.; Deng, Z.; Siciliano, M.J.
ERCC4 was previously identified in somatic cell hybrids as a human gene that corrects the nucleotide-excision-repair deficiency in mutant hamster cells. The cloning strategy for ERCC4 involved transfection of the repair-deficient hamster cell line UV41 with a human sCos-1 cosmid library derived from chromosome 16. Enhanced UV resistance was seen with one cosmid-library transformant and two secondary transformants of UV41. Cosmid clones carrying a functional ERCC4 gene were isolated from a library of a second transformant by selecting in Escherichia coli for expression of a linked neomycin-resistance gene that was present in the sCos-1 vector. The cosmids mapped to 16p13.13-p13.2, the location assigned to ERCC4 by using somatic cell hybrids. Upon transfection into UV41, six cosmid clones gave partial correction ranging from 30% to 64%, although all appeared to contain the complete gene. The capacity for in vitro excision of thymine dimers from a plasmid by transformant cell extracts correlated qualitatively with enhanced UV resistance.
Transcriptional and post-transcriptional regulation of nucleotide excision repair genes in human cells.
Lefkofsky, Hailey B; Veloso, Artur; Ljungman, Mats
Nucleotide excision repair (NER) removes DNA helix-distorting lesions induced by UV light and various chemotherapeutic agents such as cisplatin. These lesions efficiently block the elongation of transcription and need to be rapidly removed by transcription-coupled NER (TC-NER) to avoid the induction of apoptosis. Twenty-nine genes have been classified to code for proteins participating in nucleotide excision repair (NER) in human cells. Here we explored the transcriptional and post-transcriptional regulation of these NER genes across 13 human cell lines using Bru-seq and BruChase-seq, respectively. Many NER genes are relatively large in size and therefore will be easily inactivated by UV-induced transcription-blocking lesions. Furthermore, many of these genes produce transcripts that are rather unstable. Thus, these genes are expected to rapidly lose expression leading to a diminished function of NER. One such gene is ERCC6 that codes for the CSB protein critical for TC-NER. Due to its large gene size and high RNA turnover rate, the ERCC6 gene may act as dosimeter of DNA damage so that at high levels of damage, ERCC6 RNA levels would be diminished leading to the loss of CSB expression, inhibition of TC-NER and the promotion of cell death. PMID:26255935
Cloning and molecular characterization of the Chinese hamster ERCC2 nucleotide excision repair gene
Kirchner, J.M.; Salazar, E.P.; Lamerdin, J.E.
The Chinese hamster ERCC2 nucleotide excision repair gene, encoding a presumed ATP-dependent DNA helicase, was cloned from the V79 cell line, and its nucleotide sequence was determined. The {approximately}15-kb gene comprises 23 exons with a 2283-base open reading frame. The predicted 760-amino-acid protein is 98% identical to the human ERCC2/EXP (760 amino acids), 51% identical to the Saccharomyces cerevisiae RAD3 (778 amino acids), and 54% identical to the Schizosaccharomyces pombe rad15 (772 amino acids) proteins. The promoter region of the hamster ERCC2 gene contains a pyrimidine-rich stretch (42 nucleotides, 88% C+T) similar to sequences found in the promoter regions of two other nucleotide excision repair genes, a GC box, a putative {alpha}-Pal transcription factor binding site, and two CAAT boxes. There is no apparent TAATA box. No consensus polyadenylation sequence (AATAAA or its variants) was found with 663 bases 3{prime} of the translation termination codon. 54 refs., 2 figs., 2 tabs.
Transcriptional and Post-Transcriptional Regulation of Nucleotide Excision Repair Genes in Human Cells
Lefkofsky, Hailey B.; Veloso, Artur; Ljungman, Mats
Nucleotide excision repair (NER) removes DNA helix-distorting lesions induced by UV light and various chemotherapeutic agents such as cisplatin. These lesions efficiently block the elongation of transcription and need to be rapidly removed by transcription-coupled NER (TC-NER) to avoid the induction of apoptosis. Twenty-nine genes have been classified to code for proteins participating in nucleotide excision repair (NER) in human cells. Here we explored the transcriptional and post-transcriptional regulation of these NER genes across 13 human cell lines using Bru-seq and BruChase-seq, respectively. Many NER genes are relatively large in size and therefore will be easily inactivated by UV-induced transcription-blocking lesions. Furthermore, many of these genes produce transcripts that are rather unstable. Thus, these genes are expected to rapidly lose expression leading to a diminished function of NER. One such gene is ERCC6 that codes for the CSB protein critical for TC-NER. Due to its large gene size and high RNA turnover rate, the ERCC6 gene may act as dosimeter of DNA damage so that at high levels of damage, ERCC6 RNA levels would be diminished leading to the loss of CSB expression, inhibition of TC-NER and the promotion of cell death. PMID:26255935
Nucleotide excision repair of the 5 S ribosomal RNA gene assembled into a nucleosome.
Liu, X; Smerdon, M J
A-175-base pair fragment containing the Xenopus borealis somatic 5 S ribosomal RNA gene was used as a model system to determine the effect of nucleosome assembly on nucleotide excision repair (NER) of the major UV photoproduct (cyclobutane pyrimidine dimer (CPD)) in DNA. Xenopus oocyte nuclear extracts were used to carry out repair in vitro on reconstituted, positioned 5 S rDNA nucleosomes. Nucleosome structure strongly inhibits NER at many CPD sites in the 5 S rDNA fragment while having little effect at a few sites. The time course of CPD removal at 35 different sites indicates that >85% of the CPDs in the naked DNA fragment have t(12) values <2 h, whereas <26% of the t(12) values in nucleosomes are <2 h, and 15% are >8 h. Moreover, removal of histone tails from these mononucleosomes has little effect on the repair rates. Finally, nucleosome inhibition of repair shows no correlation with the rotational setting of a 14-nucleotide-long pyrimidine tract located 30 base pairs from the nucleosome dyad. These results suggest that inhibition of NER by mononucleosomes is not significantly influenced by the rotational orientation of CPDs on the histone surface, and histone tails play little (or no) role in this inhibition. PMID:10821833
TGF-β signaling links E-cadherin loss to suppression of nucleotide excision repair.
Qiang, L; Shah, P; Barcellos-Hoff, M H; He, Y Y
E-cadherin is a cell adhesion molecule best known for its function in suppressing tumor progression and metastasis. Here we show that E-cadherin promotes nucleotide excision repair through positively regulating the expression of xeroderma pigmentosum complementation group C (XPC) and DNA damage-binding protein 1 (DDB1). Loss of E-cadherin activates the E2F4 and p130/107 transcription repressor complexes to suppress the transcription of both XPC and DDB1 through activating the transforming growth factor-β (TGF-β) pathway. Adding XPC or DDB1, or inhibiting the TGF-β pathway, increases the repair of ultraviolet (UV)-induced DNA damage in E-cadherin-inhibited cells. In the mouse skin and skin tumors, UVB radiation downregulates E-cadherin. In sun-associated premalignant and malignant skin neoplasia, E-cadherin is downregulated in association with reduced XPC and DDB1 levels. These findings demonstrate a crucial role of E-cadherin in efficient DNA repair of UV-induced DNA damage, identify a new link between epithelial adhesion and DNA repair and suggest a mechanistic link of early E-cadherin loss in tumor initiation. PMID:26477308
The PARP inhibitor Olaparib disrupts base excision repair of 5-aza-2'-deoxycytidine lesions.
Orta, Manuel Luis; Höglund, Andreas; Calderón-Montaño, José Manuel; Domínguez, Inmaculada; Burgos-Morón, Estefanía; Visnes, Torkild; Pastor, Nuria; Ström, Cecilia; López-lázaro, Miguel; Helleday, Thomas
Decitabine (5-aza-2'-deoxycytidine, 5-azadC) is used in the treatment of Myelodysplatic syndrome (MDS) and Acute Myeloid Leukemia (AML). Its mechanism of action is thought to involve reactivation of genes implicated in differentiation and transformation, as well as induction of DNA damage by trapping DNA methyltranferases (DNMT) to DNA. We demonstrate for the first time that base excision repair (BER) recognizes 5-azadC-induced lesions in DNA and mediates repair. We find that BER (XRCC1) deficient cells are sensitive to 5-azadC and display an increased amount of DNA single- and double-strand breaks. The XRCC1 protein co-localizes with DNMT1 foci after 5-azadC treatment, suggesting a novel and specific role of XRCC1 in the repair of trapped DNMT1. 5-azadC-induced DNMT foci persist in XRCC1 defective cells, demonstrating a role for XRCC1 in repair of 5-azadC-induced DNA lesions. Poly (ADP-ribose) polymerase (PARP) inhibition prevents XRCC1 relocation to DNA damage sites, disrupts XRCC1-DNMT1 co-localization and thereby efficient BER. In a panel of AML cell lines, combining 5-azadC and Olaparib cause synthetic lethality. These data suggest that PARP inhibitors can be used in combination with 5-azadC to improve treatment of MDS and AML. PMID:25074383
UV sensitivity and impaired nucleotide excision repair in DNA-dependent protein kinase mutant cells.
Muller, C; Calsou, P; Frit, P; Cayrol, C; Carter, T; Salles, B
DNA-dependent protein kinase (DNA-PK), a member of the phosphatidyl-inositol (PI)3-kinase family, is involved in the repair of DNA double-strand breaks. Its regulatory subunit, Ku, binds to DNA and recruits the kinase catalytic subunit (DNA-PKcs). We show here a new role of DNA-PK in the modulation of the process of nucleotide excision repair (NER) in vivo since, as compared with their respective parental cell lines, DNA-PK mutants (scid , V-3 and xrs 6 cells) exhibit sensitivity to UV-C irradiation (2.0- to 2.5-fold) and cisplatin ( approximately 3- to 4-fold) associated with a decreased activity (40-55%) of unscheduled DNA synthesis after UV-C irradiation. Moreover, we observed that wortmannin sensitized parental cells in vivo when combined with either cisplatin or UV-C light, but had no effect on the DNA-PKcs deficient scid cells. Despite a lower repair synthesis activity (approximately 2-fold) measured in vitro with nuclear cell extracts from DNA-PK mutants, a direct involvement of DNA-PK in the NER reaction in vitro has not been observed. This study establishes a regulatory function of DNA-PK in the NER process in vivo but rules out a physical role of the complex in the repair machinery at the site of the DNA lesion. PMID:9490781
Nucleotide Excision Repair and Transcription-coupled DNA Repair Abrogate the Impact of DNA Damage on Transcription*
Nadkarni, Aditi; Burns, John A.; Gandolfi, Alberto; Chowdhury, Moinuddin A.; Cartularo, Laura; Berens, Christian; Geacintov, Nicholas E.; Scicchitano, David A.
DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N6-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N6-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N6-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N6-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N6-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER. PMID:26559971
Recruitment of the Nucleotide Excision Repair Endonuclease XPG to Sites of UV-Induced DNA Damage Depends on Functional TFIIH▿
Zotter, Angelika; Luijsterburg, Martijn S.; Warmerdam, Daniël O.; Ibrahim, Shehu; Nigg, Alex; van Cappellen, Wiggert A.; Hoeijmakers, Jan H. J.; van Driel, Roel; Vermeulen, Wim; Houtsmuller, Adriaan B.
The structure-specific endonuclease XPG is an indispensable core protein of the nucleotide excision repair (NER) machinery. XPG cleaves the DNA strand at the 3′ side of the DNA damage. XPG binding stabilizes the NER preincision complex and is essential for the 5′ incision by the ERCC1/XPF endonuclease. We have studied the dynamic role of XPG in its different cellular functions in living cells. We have created mammalian cell lines that lack functional endogenous XPG and stably express enhanced green fluorescent protein (eGFP)-tagged XPG. Life cell imaging shows that in undamaged cells XPG-eGFP is uniformly distributed throughout the cell nucleus, diffuses freely, and is not stably associated with other nuclear proteins. XPG is recruited to UV-damaged DNA with a half-life of 200 s and is bound for 4 min in NER complexes. Recruitment requires functional TFIIH, although some TFIIH mutants allow slow XPG recruitment. Remarkably, binding of XPG to damaged DNA does not require the DDB2 protein, which is thought to enhance damage recognition by NER factor XPC. Together, our data present a comprehensive view of the in vivo behavior of a protein that is involved in a complex chromatin-associated process. PMID:17000769
Genetic characterization of the nucleotide excision repair system of Neisseria gonorrhoeae.
LeCuyer, Brian E; Criss, Alison K; Seifert, H Steven
Nucleotide excision repair (NER) is universally used to recognize and remove many types of DNA damage. In eubacteria, the NER system typically consists of UvrA, UvrB, UvrC, the UvrD helicase, DNA polymerase I, and ligase. In addition, when DNA damage blocks transcription, transcription-repair coupling factor (TRCF), the product of the mfd gene, recruits the Uvr complex to repair the damage. Previous work using selected mutants and assays have indicated that pathogenic Neisseria spp. carry a functional NER system. In order to comprehensively examine the role of NER in Neisseria gonorrhoeae DNA recombination and repair processes, the predicted NER genes (uvrA, uvrB, uvrC, uvrD, and mfd) were each disrupted by a transposon insertion, and the uvrB and uvrD mutants were complemented with a copy of each gene in an ectopic locus. Each uvr mutant strain was highly sensitive to UV irradiation and also showed sensitivity to hydrogen peroxide killing, confirming that all of the NER genes in N. gonorrhoeae are functional. The effect of RecA expression on UV survival was minor in uvr mutants but much larger in the mfd mutant. All of the NER mutants demonstrated wild-type levels of pilin antigenic variation and DNA transformation. However, the uvrD mutant exhibited higher frequencies of PilC-mediated pilus phase variation and spontaneous mutation, a finding consistent with a role for UvrD in mismatch repair. We conclude that NER functions are conserved in N. gonorrhoeae and are important for the DNA repair capabilities of this strict human pathogen. PMID:19933360
Nucleotide excision repair (NER) machinery recruitment by the transcription-repair coupling factor involves unmasking of a conserved intramolecular interface
Deaconescu, Alexandra M.; Sevostyanova, Anastasia; Artsimovitch, Irina; Grigorieff, Nikolaus
Transcription-coupled DNA repair targets DNA lesions that block progression of elongating RNA polymerases. In bacteria, the transcription-repair coupling factor (TRCF; also known as Mfd) SF2 ATPase recognizes RNA polymerase stalled at a site of DNA damage, removes the enzyme from the DNA, and recruits the Uvr(A)BC nucleotide excision repair machinery via UvrA binding. Previous studies of TRCF revealed a molecular architecture incompatible with UvrA binding, leaving its recruitment mechanism unclear. Here, we examine the UvrA recognition determinants of TRCF using X-ray crystallography of a core TRCF–UvrA complex and probe the conformational flexibility of TRCF in the absence and presence of nucleotides using small-angle X-ray scattering. We demonstrate that the C-terminal domain of TRCF is inhibitory for UvrA binding, but not RNA polymerase release, and show that nucleotide binding induces concerted multidomain motions. Our studies suggest that autoinhibition of UvrA binding in TRCF may be relieved only upon engaging the DNA damage. PMID:22331906
XRCC1 and base excision repair balance in response to nitric oxide.
Mutamba, James T; Svilar, David; Prasongtanakij, Somsak; Wang, Xiao-Hong; Lin, Ying-Chih; Dedon, Peter C; Sobol, Robert W; Engelward, Bevin P
Inflammation associated reactive oxygen and nitrogen species (RONs), including peroxynitrite (ONOO(-)) and nitric oxide (NO), create base lesions that potentially play a role in the toxicity and large genomic rearrangements associated with many malignancies. Little is known about the role of base excision repair (BER) in removing these endogenous DNA lesions. Here, we explore the role of X-ray repair cross-complementing group 1 (XRCC1) in attenuating RONs-induced genotoxicity. XRCC1 is a scaffold protein critical for BER for which polymorphisms modulate the risk of cancer. We exploited CHO and human glioblastoma cell lines engineered to express varied levels of BER proteins to study XRCC1. Cytotoxicity and the levels of DNA repair intermediates (single-strand breaks; SSB) were evaluated following exposure of the cells to the ONOO(-) donor, SIN-1, and to gaseous NO. XRCC1 null cells were slightly more sensitive to SIN-1 than wild-type cells. We used small-scale bioreactors to expose cells to NO and found that XRCC1-deficient CHO cells were not sensitive. However, using a molecular beacon assay to test lesion removal in vitro, we found that XRCC1 facilitates AAG-initiated excision of two key NO-induced DNA lesions: 1,N(6)-ethenoadenine and hypoxanthine. Furthermore, overexpression of AAG rendered XRCC1-deficient cells sensitive to NO-induced DNA damage. These results show that AAG is a key glycosylase for BER of NO-induced DNA damage and that XRCC1's role in modulating sensitivity to RONs is dependent upon the cellular level of AAG. This demonstrates the importance of considering the expression of other components of the BER pathway when evaluating the impact of XRCC1 polymorphisms on cancer risk. PMID:22041025
Nucleotide Excision Repair Is Not Induced in Human Embryonic Lung Fibroblasts Treated with Environmental Pollutants
Rossner, Pavel; Spatova, Milada; Rossnerova, Andrea; Libalova, Helena; Schmuczerova, Jana; Milcova, Alena; Topinka, Jan; Sram, Radim J.
The cellular response to genotoxic treatment depends on the cell line used. Although tumor cell lines are widely used for genotoxicity tests, the interpretation of the results may be potentially hampered by changes in cellular processes caused by malignant transformation. In our study we used normal human embryonic lung fibroblasts (HEL12469 cells) and tested their response to treatment with benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) from ambient air particles <2.5 µm (PM2.5) collected in two Czech cities differing in levels and sources of air pollution. We analyzed multiple endpoints associated with exposure to polycyclic aromatic hydrocarbons (PAHs) including the levels of bulky DNA adducts and the nucleotide excision repair (NER) response [expression of XPE, XPC and XPA genes on the level of mRNA and proteins, unscheduled DNA synthesis (UDS)]. EOMs were collected in the winter and summer of 2011 in two Czech cities with different levels and sources of air pollution. The effects of the studied compounds were analyzed in the presence (+S9) and absence (–S9) of the rat liver microsomal S9 fraction. The levels of bulky DNA adducts were highest after treatment with B[a]P, followed by winter EOMs; their induction by summer EOMs was weak. The induction of both mRNA and protein expression was observed, with the most pronounced effects after treatment with B[a]P (–S9); the response induced by EOMs from both cities and seasons was substantially weaker. The expression of DNA repair genes was not accompanied by the induction of UDS activity. In summary, our results indicate that the tested compounds induced low levels of DNA damage and affected the expression of NER genes; however, nucleotide excision repair was not induced. PMID:23894430
The role of glutathione in the regulation of nucleotide excision repair during oxidative stress.
Langie, Sabine A S; Knaapen, Ad M; Houben, Joyce M J; van Kempen, Frederik C; de Hoon, Joep P J; Gottschalk, Ralph W H; Godschalk, Roger W L; van Schooten, Frederik J
Nucleotide excision repair (NER) mainly repairs bulky DNA adducts and helix distorting lesions, but is additionally considered to be a back-up system for base excision repair to remove oxidative stress induced DNA damage. Therefore, it can be speculated that NER is up-regulated or primed by oxidative stress. Exposure of human pulmonary epithelial cells (A549) to non-toxic doses of 100muM H(2)O(2) indeed showed a 2 to 4.5-fold increase in expression of XPA, XPC, ERCC4, and ERCC5, whereas the expression of ERCC1 was 5-fold decreased. Phenotypical assessment of NER capacity (i.e. recognition and incision of benzo[a]pyrene-DNA adducts) showed a significant decrease to less than 50% after H(2)O(2) exposure, which paralleled the effects of H(2)O(2) on ERCC1 expression. To study the possible involvement of glutathione (GSH) in the regulation of NER, cells were pre-incubated with 0.5mM BSO, resulting in total GSH depletion and increased intracellular oxidative stress. In GSH-depleted cells, the down-regulation of ERCC1 expression by H(2)O(2) was completely abolished and the up-regulation of ERCC4 expression was potentiated from 2.5-fold to >10-fold. Similarly, the H(2)O(2)-induced decrease in NER capacity was absent in GSH-depleted cells. Overall, our data suggest that NER capacity as well as the expression of NER related genes can be modulated by oxidative stress. ERCC1 expression and NER capacity correlated strongly (R(2)=0.85, P<0.01) after oxidant exposure, indicating ERCC1 as a specific target for oxidative stress induced modification of NER. PMID:17207589
Base excision DNA repair in the embryonic development of the sea urchin, Strongylocentrotus intermedius.
Torgasheva, Natalya A; Menzorova, Natalya I; Sibirtsev, Yurii T; Rasskazov, Valery A; Zharkov, Dmitry O; Nevinsky, Georgy A
In actively proliferating cells, such as the cells of the developing embryo, DNA repair is crucial for preventing the accumulation of mutations and synchronizing cell division. Sea urchin embryo growth was analyzed and extracts were prepared. The relative activity of DNA polymerase, apurinic/apyrimidinic (AP) endonuclease, uracil-DNA glycosylase, 8-oxoguanine-DNA glycosylase, and other glycosylases was analyzed using specific oligonucleotide substrates of these enzymes; the reaction products were resolved by denaturing 20% polyacrylamide gel electrophoresis. We have characterized the profile of several key base excision repair activities in the developing embryos (2 blastomers to mid-pluteus) of the grey sea urchin, Strongylocentrotus intermedius. The uracil-DNA glycosylase specific activity sharply increased after blastula hatching, whereas the specific activity of 8-oxoguanine-DNA glycosylase steadily decreased over the course of the development. The AP-endonuclease activity gradually increased but dropped at the last sampled stage (mid-pluteus 2). The DNA polymerase activity was high at the first cleavage division and then quickly decreased, showing a transient peak at blastula hatching. It seems that the developing sea urchin embryo encounters different DNA-damaging factors early in development within the protective envelope and later as a free-floating larva, with hatching necessitating adaptation to the shift in genotoxic stress conditions. No correlation was observed between the dynamics of the enzyme activities and published gene expression data from developing congeneric species, S. purpuratus. The results suggest that base excision repair enzymes may be regulated in the sea urchin embryos at the level of covalent modification or protein stability. PMID:27158700
Induction of DNA polymerase beta-dependent base excision repair in response to oxidative stress in vivo.
Cabelof, Diane C; Raffoul, Julian J; Yanamadala, Sunitha; Guo, ZhongMao; Heydari, Ahmad R
Base excision repair (BER) is the DNA repair pathway primarily responsible for repairing small base modifications and abasic sites caused by normal cellular metabolism or environmental insult. Strong evidence supports the requirement of DNA polymerase beta (beta-pol) in the BER pathway involving single nucleotide gap filling DNA synthesis in mammalian systems. In this study, we examine the relationship between oxidative stress, cellular levels of beta-pol and BER to determine whether oxidizing agents can upregulate BER capacity in vivo. Intraperitoneal injection of 2-nitropropane (2-NP, 100 mg/kg), an oxidative stress-inducing agent, in C57BL/6 mice was found to generate 8-hydroxydeoxyguanosine (8-OHdG) in liver tissue (4-fold increase, P < 0.001). We also observed a 4-5-fold increase in levels of DNA single strand breaks in 2-NP treated animals. The protein level of the tumor suppressor gene, p53 was also induced in liver by 2-NP (2.1-fold, P < 0.01), indicating an induction of DNA damage. In addition, we observed a 2-3-fold increase in mutant frequency in the lacI gene after exposure to 2-NP. Interestingly, an increase in DNA damage upregulated the level of beta-pol as well as BER capacity (42%, P < 0.05). These results suggest that beta-pol and BER can be upregulated in response to oxidative stress in vivo. Furthermore, data show that heterozygous beta-pol knockout (beta-pol(+/-)) mice express higher levels of p53 in response to 2-NP as compared with wild-type littermates. While the knockout and wild-type mice display similar levels of 8-OHdG after 2-NP exposure, the beta-pol(+/-) mice exhibit a significant increase in DNA single strand breaks. These findings suggest that in mice, a reduction in beta-pol expression results in a higher accumulation of DNA damage by 2-NP, thus establishing the importance of the beta-pol-dependent BER pathway in repairing oxidative damage. PMID:12189182
Cells deficient in base-excision repair reveal cancer hallmarks originating from adjustments to genetic instability
Markkanen, Enni; Fischer, Roman; Ledentcova, Marina; Kessler, Benedikt M.; Dianov, Grigory L.
Genetic instability, provoked by exogenous mutagens, is well linked to initiation of cancer. However, even in unstressed cells, DNA undergoes a plethora of spontaneous alterations provoked by its inherent chemical instability and the intracellular milieu. Base excision repair (BER) is the major cellular pathway responsible for repair of these lesions, and as deficiency in BER activity results in DNA damage it has been proposed that it may trigger the development of sporadic cancers. Nevertheless, experimental evidence for this model remains inconsistent and elusive. Here, we performed a proteomic analysis of BER deficient human cells using stable isotope labelling with amino acids in cell culture (SILAC), and demonstrate that BER deficiency, which induces genetic instability, results in dramatic changes in gene expression, resembling changes found in many cancers. We observed profound alterations in tissue homeostasis, serine biosynthesis, and one-carbon- and amino acid metabolism, all of which have been identified as cancer cell 'hallmarks'. For the first time, this study describes gene expression changes characteristic for cells deficient in repair of endogenous DNA lesions by BER. These expression changes resemble those observed in cancer cells, suggesting that genetically unstable BER deficient cells may be a source of pre-cancerous cells. PMID:25800737
Oxidative DNA damage background estimated by a system model of base excision repair
Sokhansanj, B A; Wilson, III, D M
Human DNA can be damaged by natural metabolism through free radical production. It has been suggested that the equilibrium between innate damage and cellular DNA repair results in an oxidative DNA damage background that potentially contributes to disease and aging. Efforts to quantitatively characterize the human oxidative DNA damage background level based on measuring 8-oxoguanine lesions as a biomarker have led to estimates varying over 3-4 orders of magnitude, depending on the method of measurement. We applied a previously developed and validated quantitative pathway model of human DNA base excision repair, integrating experimentally determined endogenous damage rates and model parameters from multiple sources. Our estimates of at most 100 8-oxoguanine lesions per cell are consistent with the low end of data from biochemical and cell biology experiments, a result robust to model limitations and parameter variation. Our results show the power of quantitative system modeling to interpret composite experimental data and make biologically and physiologically relevant predictions for complex human DNA repair pathway mechanisms and capacity.
Base excision repair: NMR backbone assignments of Escherichia coli formamidopyrimidine-DNA glycosylase
Buchko, Garry W.; Wallace, Susan S.; Kennedy, Michael A.
Oxidative damage is emerging as one of the most important mechanisms responsible for mutagenesis, carcinogenesis, aging, and various diseases (Farr and Kogma, 1991). One of the potential targets for oxidation is cellular DNA. While exposure to exogenous agents, such as ionizing radiation and chemicals, contributes to damaging DNA, the most important oxidative agents are endogenous, such as the reactive free radicals produced during normal oxidative metabolism (Adelman et., 1988). To mitigate the potentially deleterious effects of oxidative DNA damage virtually all aerobic organisms have developed complex repair mechanisms (Petit and Sancar, 1999). One repair mechanism, base excision repair (BER), appears to be responsible for replacing most oxidative DNA damage (David and Williams, 1998). Formamidopyrimidine-DNA glycosylase (Fpg), a 269-residue metalloprotein with a molecular weight of 30.2 kDa, is a key BER enzyme in prokaryotes (Boiteaux et al., 1987). Substrates recognized and released by Fpg include 7,8-dihydro-8-oxoguanine (8-oxoG), 2,6 diamino-4-hydroxy-5-formamido pyrimidine (Fapy-G), the adenine equivalents 8-oxoA and Fapy-A, 5-hydroxycytosine, 5-hydroxyuracil, B ureidoisobutiric acid, and a-R-hydroxy-B-ureidoisobutiric acid (Freidberg et al., 1995). In vitro Fpg bind double-stranded DNA and performs three catalytic activities: (i) DNA glycosylase, (ii) AP lyase, and (iii) deoxyribophosphodiesterase.
Enhanced nucleotide excision repair capacity in lung cancer cells by preconditioning with DNA-damaging agents
Choi, Ji Ye; Park, Jeong-Min; Yi, Joo Mi; Leem, Sun-Hee; Kang, Tae-Hong
The capacity of tumor cells for nucleotide excision repair (NER) is a major determinant of the efficacy of and resistance to DNA-damaging chemotherapeutics, such as cisplatin. Here, we demonstrate that using lesion-specific monoclonal antibodies, NER capacity is enhanced in human lung cancer cells after preconditioning with DNA-damaging agents. Preconditioning of cells with a nonlethal dose of UV radiation facilitated the kinetics of subsequent cisplatin repair and vice versa. Dual-incision assay confirmed that the enhanced NER capacity was sustained for 2 days. Checkpoint activation by ATR kinase and expression of NER factors were not altered significantly by the preconditioning, whereas association of XPA, the rate-limiting factor in NER, with chromatin was accelerated. In preconditioned cells, SIRT1 expression was increased, and this resulted in a decrease in acetylated XPA. Inhibition of SIRT1 abrogated the preconditioning-induced predominant XPA binding to DNA lesions. Taking these data together, we conclude that upregulated NER capacity in preconditioned lung cancer cells is caused partly by an increased level of SIRT1, which modulates XPA sensitivity to DNA damage. This study provides some insights into the molecular mechanism of chemoresistance through acquisition of enhanced DNA repair capacity in cancer cells. PMID:26317794
Regulation of nucleotide excision repair activity by transcriptional and post-transcriptional control of the XPA protein.
Kang, Tae-Hong; Reardon, Joyce T; Sancar, Aziz
The XPA (Xeroderma pigmentosum A) protein is one of the six core factors of the human nucleotide excision repair system. In this study we show that XPA is a rate-limiting factor in all human cell lines tested, including a normal human fibroblast cell line. The level of XPA is controlled at the transcriptional level by the molecular circadian clock and at the post-translational level by a HECT domain family E3 ubiquitin ligase called HERC2. Stabilization of XPA by downregulation of HERC2 moderately enhances excision repair activity. Conversely, downregulation of XPA by siRNA reduces excision repair activity in proportion to the level of XPA. Ubiquitination and proteolysis of XPA are inhibited by DNA damage that promotes tight association of the protein with chromatin and its dissociation from the HERC2 E3 ligase. Finally, in agreement with a recent report, we find that XPA is post-translationally modified by acetylation. However, contrary to the previous claim, we find that in mouse liver only a small fraction of XPA is acetylated and that downregulation of SIRT1 deacetylase in two human cell lines does not affect the overall repair rate. Collectively, the data reveal that XPA is a limiting factor in excision repair and that its level is coordinately regulated by the circadian clock, the ubiquitin-proteasome system and DNA damage. PMID:21193487
1999 Gordon Research Conference on Mammalian DNA Repair. Final Progress Report
This Conference will examine DNA repair as the key component in genomic surveillance that is so crucial to the overall integrity and function of mammalian cells. Recent discoveries have catapulted the field of DNA repair into a pivotal position for fundamental investigations into oncology, aging, environmental health, and developmental biology. We hope to highlight the most promising and exciting avenues of research in robust discussions at this conference. This Mammalian DNA Repair Gordon Conference differs from the past conferences in this series, in which the programs were broader in scope, with respect to topics and biological systems covered. A conference sponsored by the Genetics Society in April 1998 emphasized recombinational mechanisms for double-strand break repair and the role of mismatch repair deficiency in colorectal cancer. These topics will therefore receive somewhat less emphasis in the upcoming Conference. In view of the recent mechanistic advances in mammalian DNA repair, an upcoming comprehensive DNA repair meeting next autumn at Hilton Head; and the limited enrollment for Gordon Conferences we have decided to focus session-by-session on particular areas of controversy and/or new developments specifically in mammalian systems. Thus, the principal presentations will draw upon results from other cellular systems only to the extent that they impact our understanding of mammalian DNA repair.
An interaction between the DNA repair factor XPA and replication protein A appears essential for nucleotide excision repair.
Li, L; Lu, X; Peterson, C A; Legerski, R J
Replication protein A (RPA) is required for simian virus 40-directed DNA replication in vitro and for nucleotide excision repair (NER). Here we report that RPA and the human repair protein XPA specifically interact both in vitro and in vivo. Mapping of the RPA-interactive domains in XPA revealed that both of the largest subunits of RPA, RPA-70 and RPA-34, interact with XPA at distinct sites. A domain involved in mediating the interaction with RPA-70 was located between XPA residues 153 and 176. Deletion of highly conserved motifs within this region identified two mutants that were deficient in binding RPA in vitro and highly defective in NER both in vitro and in vivo. A second domain mediating the interaction with RPA-34 was identified within the first 58 residues in XPA. Deletion of this region, however, only moderately affects the complementing activity of XPA in vivo. Finally, the XPA-RPA complex is shown to have a greater affinity for damaged DNA than XPA alone. Taken together, these results indicate that the interaction between XPA and RPA is required for NER but that only the interaction with RPA-70 is essential. PMID:7565690
R152C DNA Pol β mutation impairs base excision repair and induces cellular transformation
Zhao, Jing; Sun, Hongfang; Zhou, Xiaolong; Wu, Xuping; He, Lingfeng; Hu, Zhigang; Chen, Haoyan; Shen, Binghui; Guo, Zhigang
DNA polymerase β (Pol β) is a key enzyme in DNA base excision repair (BER), a pathway that maintains genome integrity and stability. Pol β mutations have been detected in various types of cancers, suggesting a possible linkage between Pol β mutations and cancer. However, it is not clear whether and how Pol β mutations cause cancer onset and progression. In the current work, we show that a substitution mutation, R152C, impairs Pol β polymerase activity and BER efficiency. Cells harboring Pol β R152C are sensitive to the DNA damaging agents methyl methanesulfonate (MMS) and H2O2. Moreover, the mutant cells display a high frequency of chromatid breakages and aneuploidy and also form foci. Taken together, our data indicate that Pol β R152C can drive cellular transformation. PMID:26760506
Enzymatic Activity Assays for Base Excision Repair Enzymes in Cell Extracts from Vertebrate Cells
Çağlayan, Melike; Horton, Julie K.; Wilson, Samuel H.
We previously reported enzymatic activity assays for the base excision repair (BER) enzymes DNA polymerase β (pol β), aprataxin (APTX), and flap endonuclease 1 (FEN1) in cell extracts from Saccharomyces cerevisiae (Çağlayan and Wilson, 2014). Here, we describe a method to prepare cell extracts from vertebrate cells to investigate these enzymatic activities for the processing of the 5′-adenylated-sugar phosphate-containing BER intermediate. This new protocol complements our previous publication. The cell lines used are wild-type and APTX-deficient human lymphoblast cells from an Ataxia with Oculomotor Apraxia Type 1 (AOA1) disease patient, wild-type and APTX-null DT40 chicken B cells, and mouse embryonic fibroblast (MEF) cells. This protocol is a quick and efficient way to make vertebrate cell extracts without using commercial kits. PMID:27390764
Structure of UvrA nucleotide excision repair protein in complex with modified DNA
Jaciuk, Marcin; Nowak, Elżbieta; Skowronek, Krzysztof; Tańska, Anna; Nowotny, Marcin
One of the primary pathways for removal of DNA damage is nucleotide excision repair (NER). In bacteria, the UvrA protein is the component of NER that locates the lesion. A notable feature of NER is its ability to act on many DNA modifications that vary in chemical structure. So far, the mechanism underlying this broad specificity has been unclear. Here, we report the first crystal structure of a UvrA protein in complex with a chemically modified oligonucleotide. The structure shows that the UvrA dimer does not contact the site of lesion directly, but rather binds the DNA regions on both sides of the modification. The DNA region harboring the modification is deformed, with the double helix bent and unwound. UvrA uses damage-induced deformations of the DNA and a less rigid structure of the modified double helix for indirect readout of the lesion. PMID:21240268
Nucleotide excision repair in chromatin: the shape of things to come.
Reed, Simon H
Much of our mechanistic understanding of nucleotide excision repair (NER) has been derived from biochemical studies that have analysed the reaction as it occurs on DNA substrates that are not representative of DNA as it exists in the living cell. These studies have been extremely useful in deciphering the core mechanism of the NER reaction, but efforts to understand how NER operates in chromatin have been hampered in part because assembling DNA into nucleosomes, the first level of chromatin compaction, is inhibitory to NER in vitro. However, recent research using biochemical, genetic and cell-based studies is now providing us with the first insights into the molecular mechanism of NER as it occurs in the cellular context. A number of recent studies have provided glimpses of a chromatin--NER connection. Here I review this literature and evaluate how it might aid our understanding, and shape our future research into NER. PMID:15905137
UVSSA and USP7: new players regulating transcription-coupled nucleotide excision repair in human cells
Transcription-coupled nucleotide excision repair (TC-NER) specifically removes DNA damage located in actively transcribed genes. Defects in TC-NER are associated with several human disorders, including Cockayne syndrome (CS) and ultraviolet (UV)-sensitive syndrome (UVSS). Using exome sequencing, and genetic and proteomic approaches, three recent studies have identified mutations in the UVSSA gene as being responsible for UVSS-A. These findings suggest a new mechanistic model involving UV-stimulated scaffold protein A (UVSSA) and the ubiquitin-specific protease 7 (USP7) in the fate of stalled RNA polymerase II during TC-NER, and provide insights into the diverse clinical features of CS and UVSS. PMID:22621766
UVSSA and USP7: new players regulating transcription-coupled nucleotide excision repair in human cells.
Sarasin, Alain
Transcription-coupled nucleotide excision repair (TC-NER) specifically removes DNA damage located in actively transcribed genes. Defects in TC-NER are associated with several human disorders, including Cockayne syndrome (CS) and ultraviolet (UV)-sensitive syndrome (UVSS). Using exome sequencing, and genetic and proteomic approaches, three recent studies have identified mutations in the UVSSA gene as being responsible for UVSS-A. These findings suggest a new mechanistic model involving UV-stimulated scaffold protein A (UVSSA) and the ubiquitin-specific protease 7 (USP7) in the fate of stalled RNA polymerase II during TC-NER, and provide insights into the diverse clinical features of CS and UVSS. PMID:22621766
Nucleotide excision repair pathway assessment in DNA exposed to low-intensity red and infrared lasers
Fonseca, A.S.; Campos, V.M.A.; Magalhães, L.A.G.; Paoli, F.
Low-intensity lasers are used for prevention and management of oral mucositis induced by anticancer therapy, but the effectiveness of treatment depends on the genetic characteristics of affected cells. This study evaluated the survival and induction of filamentation of Escherichia coli cells deficient in the nucleotide excision repair pathway, and the action of T4endonuclease V on plasmid DNA exposed to low-intensity red and near-infrared laser light. Cultures of wild-type (strain AB1157) E. coli and strain AB1886 (deficient in uvrA protein) were exposed to red (660 nm) and infrared (808 nm) lasers at various fluences, powers and emission modes to study bacterial survival and filamentation. Also, plasmid DNA was exposed to laser light to study DNA lesions produced in vitro by T4endonuclease V. Low-intensity lasers:i) had no effect on survival of wild-type E. coli but decreased the survival of uvrA protein-deficient cells,ii) induced bacterial filamentation, iii) did not alter the electrophoretic profile of plasmids in agarose gels, andiv) did not alter the electrophoretic profile of plasmids incubated with T4 endonuclease V. These results increase our understanding of the effects of laser light on cells with various genetic characteristics, such as xeroderma pigmentosum cells deficient in nucleotide excision pathway activity in patients with mucositis treated by low-intensity lasers. PMID:26445337
Nucleotide excision repair pathway assessment in DNA exposed to low-intensity red and infrared lasers.
Fonseca, A S; Campos, V M A; Magalhães, L A G; Paoli, F
Low-intensity lasers are used for prevention and management of oral mucositis induced by anticancer therapy, but the effectiveness of treatment depends on the genetic characteristics of affected cells. This study evaluated the survival and induction of filamentation of Escherichia coli cells deficient in the nucleotide excision repair pathway, and the action of T4endonuclease V on plasmid DNA exposed to low-intensity red and near-infrared laser light. Cultures of wild-type (strain AB1157) E. coli and strain AB1886 (deficient in uvrA protein) were exposed to red (660 nm) and infrared (808 nm) lasers at various fluences, powers and emission modes to study bacterial survival and filamentation. Also, plasmid DNA was exposed to laser light to study DNA lesions produced in vitro by T4endonuclease V. Low-intensity lasers:i) had no effect on survival of wild-type E. coli but decreased the survival of uvrA protein-deficient cells,ii) induced bacterial filamentation, iii) did not alter the electrophoretic profile of plasmids in agarose gels, andiv) did not alter the electrophoretic profile of plasmids incubated with T4 endonuclease V. These results increase our understanding of the effects of laser light on cells with various genetic characteristics, such as xeroderma pigmentosum cells deficient in nucleotide excision pathway activity in patients with mucositis treated by low-intensity lasers. PMID:26445337
Nucleotide Excision Repair and Vitamin D—Relevance for Skin Cancer Therapy
Pawlowska, Elzbieta; Wysokinski, Daniel; Blasiak, Janusz
Ultraviolet (UV) radiation is involved in almost all skin cancer cases, but on the other hand, it stimulates the production of pre-vitamin D3, whose active metabolite, 1,25-dihydroxyvitamin D3 (1,25VD3), plays important physiological functions on binding with its receptor (vitamin D receptor, VDR). UV-induced DNA damages in the form of cyclobutane pyrimidine dimers or (6-4)-pyrimidine-pyrimidone photoproducts are frequently found in skin cancer and its precursors. Therefore, removing these lesions is essential for the prevention of skin cancer. As UV-induced DNA damages are repaired by nucleotide excision repair (NER), the interaction of 1,25VD3 with NER components can be important for skin cancer transformation. Several studies show that 1,25VD3 protects DNA against damage induced by UV, but the exact mechanism of this protection is not completely clear. 1,25VD3 was also shown to affect cell cycle regulation and apoptosis in several signaling pathways, so it can be considered as a potential modulator of the cellular DNA damage response, which is crucial for mutagenesis and cancer transformation. 1,25VD3 was shown to affect DNA repair and potentially NER through decreasing nitrosylation of DNA repair enzymes by NO overproduction by UV, but other mechanisms of the interaction between 1,25VD3 and NER machinery also are suggested. Therefore, the array of NER gene functioning could be analyzed and an appropriate amount of 1.25VD3 could be recommended to decrease UV-induced DNA damage important for skin cancer transformation. PMID:27058533
Nucleotide Excision Repair and Vitamin D-Relevance for Skin Cancer Therapy.
Pawlowska, Elzbieta; Wysokinski, Daniel; Blasiak, Janusz
Ultraviolet (UV) radiation is involved in almost all skin cancer cases, but on the other hand, it stimulates the production of pre-vitamin D3, whose active metabolite, 1,25-dihydroxyvitamin D3 (1,25VD3), plays important physiological functions on binding with its receptor (vitamin D receptor, VDR). UV-induced DNA damages in the form of cyclobutane pyrimidine dimers or (6-4)-pyrimidine-pyrimidone photoproducts are frequently found in skin cancer and its precursors. Therefore, removing these lesions is essential for the prevention of skin cancer. As UV-induced DNA damages are repaired by nucleotide excision repair (NER), the interaction of 1,25VD3 with NER components can be important for skin cancer transformation. Several studies show that 1,25VD3 protects DNA against damage induced by UV, but the exact mechanism of this protection is not completely clear. 1,25VD3 was also shown to affect cell cycle regulation and apoptosis in several signaling pathways, so it can be considered as a potential modulator of the cellular DNA damage response, which is crucial for mutagenesis and cancer transformation. 1,25VD3 was shown to affect DNA repair and potentially NER through decreasing nitrosylation of DNA repair enzymes by NO overproduction by UV, but other mechanisms of the interaction between 1,25VD3 and NER machinery also are suggested. Therefore, the array of NER gene functioning could be analyzed and an appropriate amount of 1.25VD3 could be recommended to decrease UV-induced DNA damage important for skin cancer transformation. PMID:27058533
Exposure of Human Lung Cells to Tobacco Smoke Condensate Inhibits the Nucleotide Excision Repair Pathway
Holcomb, Nathaniel; Goswami, Mamta; Han, Sung Gu; Clark, Samuel; Orren, David K.; Gairola, C. Gary; Mellon, Isabel
Exposure to tobacco smoke is the number one risk factor for lung cancer. Although the DNA damaging properties of tobacco smoke have been well documented, relatively few studies have examined its effect on DNA repair pathways. This is especially true for the nucleotide excision repair (NER) pathway which recognizes and removes many structurally diverse DNA lesions, including those introduced by chemical carcinogens present in tobacco smoke. The aim of the present study was to investigate the effect of tobacco smoke on NER in human lung cells. We studied the effect of cigarette smoke condensate (CSC), a surrogate for tobacco smoke, on the NER pathway in two different human lung cell lines; IMR-90 lung fibroblasts and BEAS-2B bronchial epithelial cells. To measure NER, we employed a slot-blot assay to quantify the introduction and removal of UV light-induced 6–4 photoproducts and cyclobutane pyrimidine dimers. We find a dose-dependent inhibition of 6–4 photoproduct repair in both cell lines treated with CSC. Additionally, the impact of CSC on the abundance of various NER proteins and their respective RNAs was investigated. The abundance of XPC protein, which is required for functional NER, is significantly reduced by treatment with CSC while the abundance of XPA protein, also required for NER, is unaffected. Both XPC and XPA RNA levels are modestly reduced by CSC treatment. Finally, treatment of cells with MG-132 abrogates the reduction in the abundance of XPC protein produced by treatment with CSC, suggesting that CSC enhances proteasome-dependent turnover of the protein that is mediated by ubiquitination. Together, these findings indicate that tobacco smoke can inhibit the same DNA repair pathway that is also essential for the removal of some of the carcinogenic DNA damage introduced by smoke itself, increasing the DNA damage burden of cells exposed to tobacco smoke. PMID:27391141
Genome-wide analysis of human global and transcription-coupled excision repair of UV damage at single-nucleotide resolution
Hu, Jinchuan; Adar, Sheera; Selby, Christopher P.
We developed a method for genome-wide mapping of DNA excision repair named XR-seq (excision repair sequencing). Human nucleotide excision repair generates two incisions surrounding the site of damage, creating an ∼30-mer. In XR-seq, this fragment is isolated and subjected to high-throughput sequencing. We used XR-seq to produce stranded, nucleotide-resolution maps of repair of two UV-induced DNA damages in human cells: cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine–pyrimidone photoproducts [(6-4)PPs]. In wild-type cells, CPD repair was highly associated with transcription, specifically with the template strand. Experiments in cells defective in either transcription-coupled excision repair or general excision repair isolated the contribution of each pathway to the overall repair pattern and showed that transcription-coupled repair of both photoproducts occurs exclusively on the template strand. XR-seq maps capture transcription-coupled repair at sites of divergent gene promoters and bidirectional enhancer RNA (eRNA) production at enhancers. XR-seq data also uncovered the repair characteristics and novel sequence preferences of CPDs and (6-4)PPs. XR-seq and the resulting repair maps will facilitate studies of the effects of genomic location, chromatin context, transcription, and replication on DNA repair in human cells. PMID:25934506
Nucleotide Excision Repair Pathway Polymorphisms and Pancreatic Cancer Risk: Evidence for role of MMS19L
McWilliams, Robert R.; Bamlet, William R.; de Andrade, Mariza; Rider, David N.; Cunningham, Julie M.; Petersen, Gloria M.
Background Nucleotide excision repair (NER) is a vital response to DNA damage, including damage from tobacco exposure. Single nucleotide polymorphisms (SNPs) in the NER pathway may encode alterations that affect DNA repair function and therefore influence risk for pancreatic cancer development. Methods A clinic based case-control study in non-Hispanic white persons compared 1,143 patients with pancreatic adenocarcinoma with 1,097 healthy controls. Twenty-seven genes directly and indirectly involved in the NER pathway were identified and 236 tag-SNPs were selected from 26 of these (one had no SNPs identified). Association studies were performed at the gene level by principal components analysis, while recursive partitioning analysis was utilized to identify potential gene-gene and gene-environment interactions within the pathway. At the individual SNP level, adjusted additive, dominant, and recessive models were investigated, and gene-environment interactions were also assessed. Results Gene level analyses showed an association of MMS19L genotype (chromosome 10q24.1) with altered pancreatic cancer risk (p=0.023). Haplotype analysis of MMS19L also showed a significant association (p=0.0132). Analyses of 7 individual SNPs in this gene showed both protective and risk associations for minor alleles, broadly distributed across patient subgroups defined by smoking status, sex, and age. Conclusion In a candidate pathway SNP association study analysis, common variation in a NER gene, MMS19L, was associated with risk for pancreatic cancer. PMID:19318433
Oxidative damage to RPA limits the nucleotide excision repair capacity of human cells
Guven, Melisa; Brem, Reto; Macpherson, Peter; Peacock, Matthew; Karran, Peter
Nucleotide excision repair (NER) protects against sunlight-induced skin cancer. Defective NER is associated with photosensitivity and a high skin cancer incidence. Some clinical treatments that cause photosensitivity can also increase skin cancer risk. Among these, the immunosuppressant azathioprine and the fluoroquinolone antibiotics ciprofloxacin and ofloxacin, interact with UVA radiation to generate reactive oxygen species (ROS) that diminish NER capacity by causing protein damage. The RPA DNA binding protein plays a pivotal role in DNA metabolism and is an essential component of NER. The relationship between protein oxidation and NER inhibition was investigated in cultured human cells expressing different levels of RPA. We show here that RPA is limiting for NER and that oxidative damage to RPA compromises NER capability. Our findings reveal that cellular RPA is surprisingly vulnerable to oxidation and we identify oxidized forms of RPA that are associated with impaired NER. The vulnerability of NER to inhibition by oxidation provides a connection between cutaneous photosensitivity, protein damage and increased skin cancer risk. Our findings emphasize that damage to DNA repair proteins, as well as to DNA itself is likely to be an important contributor to skin cancer risk. PMID:26134950
Base excision repair deficient mice lacking the Aag alkyladenine DNA glycosylase
Engelward, Bevin P.; Weeda, Geert; Wyatt, Michael D.; Broekhof, José L. M.; de Wit, Jan; Donker, Ingrid; Allan, James M.; Gold, Barry; Hoeijmakers, Jan H. J.; Samson, Leona D.
3-methyladenine (3MeA) DNA glycosylases remove 3MeAs from alkylated DNA to initiate the base excision repair pathway. Here we report the generation of mice deficient in the 3MeA DNA glycosylase encoded by the Aag (Mpg) gene. Alkyladenine DNA glycosylase turns out to be the major DNA glycosylase not only for the cytotoxic 3MeA DNA lesion, but also for the mutagenic 1,N6-ethenoadenine (ɛA) and hypoxanthine lesions. Aag appears to be the only 3MeA and hypoxanthine DNA glycosylase in liver, testes, kidney, and lung, and the only ɛA DNA glycosylase in liver, testes, and kidney; another ɛA DNA glycosylase may be expressed in lung. Although alkyladenine DNA glycosylase has the capacity to remove 8-oxoguanine DNA lesions, it does not appear to be the major glycosylase for 8-oxoguanine repair. Fibroblasts derived from Aag −/− mice are alkylation sensitive, indicating that Aag −/− mice may be similarly sensitive. PMID:9371804
DNA with Damage in Both Strands as Affinity Probes and Nucleotide Excision Repair Substrates.
Lukyanchikova, N V; Petruseva, I O; Evdokimov, A N; Silnikov, V N; Lavrik, O I
Nucleotide excision repair (NER) is a multistep process of recognition and elimination of a wide spectrum of damages that cause significant distortions in DNA structure, such as UV-induced damage and bulky chemical adducts. A series of model DNAs containing new bulky fluoro-azidobenzoyl photoactive lesion dC(FAB) and well-recognized nonnucleoside lesions nFlu and nAnt have been designed and their interaction with repair proteins investigated. We demonstrate that modified DNA duplexes dC(FAB)/dG (probe I), dC(FAB)/nFlu+4 (probe II), and dC(FAB)/nFlu-3 (probe III) have increased (as compared to unmodified DNA, umDNA) structure-dependent affinity for XPC-HR23B (Kdum > KdI > KdII ≈ KdIII) and differentially crosslink to XPC and proteins of NER-competent extracts. The presence of dC(FAB) results in (i) decreased melting temperature (ΔTm = -3°C) and (ii) 12° DNA bending. The extended dC(FAB)/dG-DNA (137 bp) was demonstrated to be an effective NER substrate. Lack of correlation between the affinity to XPC-HR23B and substrate properties of the model DNA suggests a high impact of the verification stage on the overall NER process. In addition, DNAs containing closely positioned, well-recognized lesions in the complementary strands represent hardly repairable (dC(FAB)/nFlu+4, dC(FAB)/nFlu-3) or irreparable (nFlu/nFlu+4, nFlu/nFlu-3, nAnt/nFlu+4, nAnt/nFlu-3) structures. Our data provide evidence that the NER system of higher eukaryotes recognizes and eliminates damaged DNA fragments on a multi-criterion basis. PMID:27262196
Nucleotide Excision Repair Gene ERCC2 and ERCC5 Variants Increase Risk of Uterine Cervical Cancer
Joo, Jungnam; Yoon, Kyong-Ah; Hayashi, Tomonori; Kong, Sun-Young; Shin, Hye-Jin; Park, Boram; Kim, Young Min; Hwang, Sang-Hyun; Kim, Jeongseon; Shin, Aesun; Kim, Joo-Young
Purpose Defects in the DNA damage repair process can cause genomic instability and play an important role in cervical carcinogenesis. The purpose of this study was to analyze the association of 29 candidate single nucleotide polymorphisms (SNPs) in genes in the DNA repair pathway, TP53, and TP53BP1 with the risk of cervical cancer. Materials and Methods Twenty-nine SNPs in four genes in the DNA repair pathway (ERCC2, ERCC5, NBS1, and XRCC1), TP53, and TP53BP1 were genotyped for 478 cervical cancer patients and 922 healthy control subjects, and their effects on cervical carcinogenesis were analyzed. Results The most significant association was found for rs17655 in ERCC5, with an age-adjusted p-value < 0.0001, for which a strong additive effect of the risk allele C was observed (odds ratio, 2.01 for CC to GG). On the other hand, another significant polymorphism rs454421 in ERCC2 showed a dominant effect (odds ratio, 1.68 for GA+AA to GG) with an age-adjusted p-value of 0.0009. The association of these polymorphisms remained significant regardless of the age of onset. The significant result for rs17655 was also consistent for subgroups of patients defined by histology and human papillomavirus (HPV) types. However, for rs454421, the association was observed only in patients with squamous cell carcinoma and non-HPV 18 type. Conclusion The results of this study show a novel association of cervical cancer and the genes involved in the nucleotide excision pathway in the Korean population. PMID:26130668
DNA excision repair in cell extracts from human cell lines exhibiting hypersensitivity to DNA-damaging agents
Hansson, J.; Keyse, S.M.; Lindahl, T.; Wood, R.D. )
Whole cell extracts from human lymphoid cell lines can perform in vitro DNA repair synthesis in plasmids damaged by agents including UV or cis-diamminedichloroplatinum(II) (cis-DDP). Extracts from xeroderma pigmentosum (XP) cells are defective in repair synthesis. We have now studied in vitro DNA repair synthesis using extracts from lymphoblastoid cell lines representing four human hereditary syndromes with increased sensitivity to DNA-damaging agents. Extracts of cell lines from individuals with the sunlight-sensitive disorders dysplastic nevus syndrome or Cockayne's syndrome (complementation groups A and B) showed normal DNA repair synthesis in plasmids with UV photoproducts. This is consistent with in vivo measurements of the overall DNA repair capacity in such cell lines. A number of extracts were prepared from two cell lines representing the variant form of XP (XP-V). Half of the extracts prepared showed normal levels of in vitro DNA repair synthesis in plasmids containing UV lesions, but the remainder of the extracts from the same cell lines showed deficient repair synthesis, suggesting the possibility of an unusually labile excision repair protein in XP-V. Fanconi's anemia (FA) cells show cellular hypersensitivity to cross-linking agents including cis-DDP. Extracts from cell lines belonging to two different complementation groups of FA showed normal DNA repair synthesis in plasmids containing cis-DDP or UV adducts. Thus, there does not appear to be an overall excision repair defect in FA, but the data do not exclude a defect in the repair of interstrand DNA cross-links.
Nucleotide Excision Repair, Mismatch Repair, and R-Loops Modulate Convergent Transcription-Induced Cell Death and Repeat Instability
Lin, Yunfu; Wilson, John H.
Expansion of CAG•CTG tracts located in specific genes is responsible for 13 human neurodegenerative disorders, the pathogenic mechanisms of which are not yet well defined. These disease genes are ubiquitously expressed in human tissues, and transcription has been identified as one of the major pathways destabilizing the repeats. Transcription-induced repeat instability depends on transcription-coupled nucleotide excision repair (TC-NER), the mismatch repair (MMR) recognition component MSH2/MSH3, and RNA/DNA hybrids (R-loops). Recently, we reported that simultaneous sense and antisense transcription–convergent transcription–through a CAG repeat not only promotes repeat instability, but also induces a cell stress response, which arrests the cell cycle and eventually leads to massive cell death via apoptosis. Here, we use siRNA knockdowns to investigate whether NER, MMR, and R-loops also modulate convergent-transcription-induced cell death and repeat instability. We find that siRNA-mediated depletion of TC-NER components increases convergent transcription-induced cell death, as does the simultaneous depletion of RNase H1 and RNase H2A. In contrast, depletion of MSH2 decreases cell death. These results identify TC-NER, MMR recognition, and R-loops as modulators of convergent transcription-induced cell death and shed light on the molecular mechanism involved. We also find that the TC-NER pathway, MSH2, and R-loops modulate convergent transcription-induced repeat instability. These observations link the mechanisms of convergent transcription-induced repeat instability and convergent transcription-induced cell death, suggesting that a common structure may trigger both outcomes. PMID:23056461
Nonhomologous end joining of complex DNA double-strand breaks with proximal thymine glycol and interplay with base excision repair.
Almohaini, Mohammed; Chalasani, Sri Lakshmi; Bafail, Duaa; Akopiants, Konstantin; Zhou, Tong; Yannone, Steven M; Ramsden, Dale A; Hartman, Matthew C T; Povirk, Lawrence F
DNA double-strand breaks induced by ionizing radiation are often accompanied by ancillary oxidative base damage that may prevent or delay their repair. In order to better define the features that make some DSBs repair-resistant, XLF-dependent nonhomologous end joining of blunt-ended DSB substrates having the oxidatively modified nonplanar base thymine glycol at the first (Tg1), second (Tg2), third (Tg3) or fifth (Tg5) positions from one 3' terminus, was examined in human whole-cell extracts. Tg at the third position had little effect on end-joining even when present on both ends of the break. However, Tg as the terminal or penultimate base was a major barrier to end joining (>10-fold reduction in ligated products) and an absolute barrier when present at both ends. Dideoxy trapping of base excision repair intermediates indicated that Tg was excised from Tg1, Tg2 and Tg3 largely if not exclusively after DSB ligation. However, Tg was rapidly excised from the Tg5 substrate, resulting in a reduced level of DSB ligation, as well as slow concomitant resection of the opposite strand. Ligase reactions containing only purified Ku, XRCC4, ligase IV and XLF showed that ligation of Tg3 and Tg5 was efficient and only partially XLF-dependent, whereas ligation of Tg1 and Tg2 was inefficient and only detectable in the presence of XLF. Overall, the results suggest that promoting ligation of DSBs with proximal base damage may be an important function of XLF, but that Tg can still be a major impediment to repair, being relatively resistant to both trimming and ligation. Moreover, it appears that base excision repair of Tg can sometimes interfere with repair of DSBs that would otherwise be readily rejoined. PMID:27049455
Developing an in silico model of the modulation of base excision repair using methoxyamine for more targeted cancer therapeutics.
Gurkan-Cavusoglu, Evren; Avadhani, Sriya; Liu, Lili; Kinsella, Timothy J; Loparo, Kenneth A
Base excision repair (BER) is a major DNA repair pathway involved in the processing of exogenous non-bulky base damages from certain classes of cancer chemotherapy drugs as well as ionising radiation (IR). Methoxyamine (MX) is a small molecule chemical inhibitor of BER that is shown to enhance chemotherapy and/or IR cytotoxicity in human cancers. In this study, the authors have analysed the inhibitory effect of MX on the BER pathway kinetics using a computational model of the repair pathway. The inhibitory effect of MX depends on the BER efficiency. The authors have generated variable efficiency groups using different sets of protein concentrations generated by Latin hypercube sampling, and they have clustered simulation results into high, medium and low efficiency repair groups. From analysis of the inhibitory effect of MX on each of the three groups, it is found that the inhibition is most effective for high efficiency BER, and least effective for low efficiency repair. PMID:23847811
Molecular analysis of plasmid DNA repair within ultraviolet-irradiated Escherichia coli. II. UvrABC-initiated excision repair and photolyase-catalyzed dimer monomerization
Gruskin, E.A.; Lloyd, R.S.
In this study, a novel approach to the analysis of DNA repair in Escherichia coli was employed which allowed the first direct determination of the mechanisms by which endogenous DNA repair enzymes encounter target sites in vivo. An in vivo plasmid DNA repair analysis was employed to discriminate between two possible mechanisms of target site location: a processive DNA scanning mechanism or a distributive random diffusion mechanism. The results demonstrate that photolyase acts by a distributive mechanism within E. coli. In contrast, UvrABC-initiated excision repair occurs by a limited processive DNA scanning mechanism. A majority of the dimer sites on a given plasmid molecule were repaired prior to the dissociation of the UvrABC complex. Furthermore, plasmid DNA repair catalyzed by the UvrABC complex occurs without a detectable accumulation of nicked plasmid intermediates despite the fact that the UvrABC complex generates dual incisions in the DNA at the site of a pyrimidine dimer. Therefore, the binding or assembly of the UvrABC complex on DNA at the site of a pyrimidine dimer represents the rate-limiting step in the overall process of UvrABC-initiated excision repair in vivo.
Molecular cloning and 3D structure modeling of APEX1, DNA base excision repair enzyme from the Camel, Camelus dromedarius.
Ataya, Farid Shokry; Fouad, Dalia; Malik, Ajamaluddin; Saeed, Hesham Mahmoud
The domesticated one-humped camel, Camelus dromedarius, is one of the most important animals in the Arabian Desert. It is exposed most of its life to both intrinsic and extrinsic genotoxic factors that are known to cause gross DNA alterations in many organisms. Ionic radiation and sunlight are known producers of Reactive Oxygen Species (ROS), one of the causes for DNA lesions. The damaged DNA is repaired by many enzymes, among of them Base Excision Repair enzymes, producing the highly mutagenic apurinic/apyrimidinicsites (AP sites). Therefore, recognition of AP sites is fundamental to cell/organism survival. In the present work, the full coding sequence of a putative cAPEX1 gene was amplified for the first time from C. dromedarius by RT-PCR and cloned (NCBI accession number are HM209828 and ADJ96599 for nucleotides and amino acids, respectively). cDNA sequencing was deduced to be 1041 nucleotides, of which 954 nucleotides encode a protein of 318 amino acids, similar to the coding region of the APEX1 gene and the protein from many other species. The calculated molecular weight and isoelectric point of cAPEX1 using Bioinformatics tools was 35.5 kDa and 8.11, respectively. The relative expressions of cAPEX1 in camel kidney, spleen, lung and testis were examined using qPCR and compared with that of the liver using a 18S ribosomal subunit as endogenous control. The highest level of cAPEX1 transcript was found in the testis; 325% higher than the liver, followed by spleen (87%), kidney (20%) and lung (5%), respectively. The cAPEX1 is 94%-97% similar to their mammalian counterparts. Phylogenetic analysis revealed that cAPEX1 is grouped together with that of S. scrofa. The predicted 3D structure of cAPEX1 has similar folds and topology with the human (hAPEX1). The root-mean-square deviation (rmsd) between cAPEX1 and hAPEX1 was 0.582 and the Q-score was 0.939. PMID:22942721
ERCC4 (XPF) encodes a human nucleotide excision repair protein with eukaryotic recombination homologs.
Brookman, K W; Lamerdin, J E; Thelen, M P; Hwang, M; Reardon, J T; Sancar, A; Zhou, Z Q; Walter, C A; Parris, C N; Thompson, L H
ERCC4 is an essential human gene in the nucleotide excision repair (NER) pathway, which is responsible for removing UV-C photoproducts and bulky adducts from DNA. Among the NER genes, ERCC4 and ERCC1 are also uniquely involved in removing DNA interstrand cross-linking damage. The ERCC1-ERCC4 heterodimer, like the homologous Rad10-Rad1 complex, was recently found to possess an endonucleolytic activity that incises on the 5' side of damage. The ERCC4 gene, assigned to chromosome 16p13.1-p13.2, was previously isolated by using a chromosome 16 cosmid library. It corrects the defect in Chinese hamster ovary (CHO) mutants of NER complementation group 4 and is implicated in complementation group F of the human disorder xeroderma pigmentosum. We describe the ERCC4 gene structure and functional cDNA sequence encoding a 916-amino-acid protein (104 kDa), which has substantial homology with the eukaryotic DNA repair and recombination proteins MEI-9 (Drosophila melanogaster), Rad16 (Schizosaccharomyces pombe), and Rad1 (Saccharomyces cerevisiae). ERCC4 cDNA efficiently corrected mutants in rodent NER complementation groups 4 and 11, showing the equivalence of these groups, and ERCC4 protein levels were reduced in mutants of both groups. In cells of an XP-F patient, the ERCC4 protein level was reduced to less than 5%, consistent with XPF being the ERCC4 gene. The considerable identity (40%) between ERCC4 and MEI-9 suggests a possible involvement of ERCC4 in meiosis. In baboon tissues, ERCC4 was expressed weakly and was not significantly higher in testis than in nonmeiotic tissues. PMID:8887684
Role of the Escherichia coli nucleotide excision repair proteins in DNA replication.
Moolenaar, G F; Moorman, C; Goosen, N
DNA polymerase I (PolI) functions both in nucleotide excision repair (NER) and in the processing of Okazaki fragments that are generated on the lagging strand during DNA replication. Escherichia coli cells completely lacking the PolI enzyme are viable as long as they are grown on minimal medium. Here we show that viability is fully dependent on the presence of functional UvrA, UvrB, and UvrD (helicase II) proteins but does not require UvrC. In contrast, delta polA cells grow even better when the uvrC gene has been deleted. Apparently UvrA, UvrB, and UvrD are needed in a replication backup system that replaces the PolI function, and UvrC interferes with this alternative replication pathway. With specific mutants of UvrC we could show that the inhibitory effect of this protein is related to its catalytic activity that on damaged DNA is responsible for the 3' incision reaction. Specific mutants of UvrA and UvrB were also studied for their capacity to support the PolI-independent replication. Deletion of the UvrC-binding domain of UvrB resulted in a phenotype similar to that caused by deletion of the uvrC gene, showing that the inhibitory incision activity of UvrC is mediated via binding to UvrB. A mutation in the N-terminal zinc finger domain of UvrA does not affect NER in vivo or in vitro. The same mutation, however, does give inviability in combination with the delta polA mutation. Apparently the N-terminal zinc-binding domain of UvrA has specifically evolved for a function outside DNA repair. A model for the function of the UvrA, UvrB, and UvrD proteins in the alternative replication pathway is discussed. PMID:11004168
Base excision repair enzymes protect abasic sites in duplex DNA from interstrand cross-links.
Admiraal, Suzanne J; O'Brien, Patrick J
Hydrolysis of the N-glycosyl bond between a nucleobase and deoxyribose leaves an abasic site within duplex DNA. The abasic site can react with exocyclic amines of nucleobases on the complementary strand to form interstrand DNA-DNA cross-links (ICLs). We find that several enzymes from the base excision repair (BER) pathway protect an abasic site on one strand of a DNA duplex from cross-linking with an amine on the opposing strand. Human alkyladenine DNA glycosylase (AAG) and Escherichia coli 3-methyladenine DNA glycosylase II (AlkA) accomplish this by binding tightly to the abasic site and sequestering it. AAG protects an abasic site opposite T, the product of its canonical glycosylase reaction, by a factor of ∼10-fold, as estimated from its inhibition of the reaction of an exogenous amine with the damaged DNA. Human apurinic/apyrimidinic site endonuclease 1 and E. coli endonuclease III both decrease the amount of ICL at equilibrium by generating a single-strand DNA nick at the abasic position as it is liberated from the cross-link. The reversibility of the reaction between amines and abasic sites allows BER enzymes to counter the potentially disruptive effects of this type of cross-link on DNA transactions. PMID:25679877
Genome Instability in Development and Aging: Insights from Nucleotide Excision Repair in Humans, Mice, and Worms
Edifizi, Diletta; Schumacher, Björn
DNA damage causally contributes to aging and cancer. Congenital defects in nucleotide excision repair (NER) lead to distinct cancer-prone and premature aging syndromes. The genetics of NER mutations have provided important insights into the distinct consequences of genome instability. Recent work in mice and C. elegans has shed new light on the mechanisms through which developing and aging animals respond to persistent DNA damage. The various NER mouse mutants have served as important disease models for Xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD), while the traceable genetics of C. elegans have allowed the mechanistic delineation of the distinct outcomes of genome instability in metazoan development and aging. Intriguingly, highly conserved longevity assurance mechanisms respond to transcription-blocking DNA lesions in mammals as well as in worms and counteract the detrimental consequences of persistent DNA damage. The insulin-like growth factor signaling (IIS) effector transcription factor DAF-16 could indeed overcome DNA damage-driven developmental growth delay and functional deterioration even when DNA damage persists. Longevity assurance mechanisms might thus delay DNA damage-driven aging by raising the threshold when accumulating DNA damage becomes detrimental for physiological tissue functioning. PMID:26287260
Conservation of the Nucleotide Excision Repair Pathway: Characterization of Hydra Xeroderma Pigmentosum Group F Homolog
Barve, Apurva; Ghaskadbi, Saroj; Ghaskadbi, Surendra
Hydra, one of the earliest metazoans with tissue grade organization and nervous system, is an animal with a remarkable regeneration capacity and shows no signs of organismal aging. We have for the first time identified genes of the nucleotide excision repair (NER) pathway from hydra. Here we report cloning and characterization of hydra homolog of xeroderma pigmentosum group F (XPF) gene that encodes a structure-specific 5′ endonuclease which is a crucial component of NER. In silico analysis shows that hydra XPF amino acid sequence is very similar to its counterparts from other animals, especially vertebrates, and shows all features essential for its function. By in situ hybridization, we show that hydra XPF is expressed prominently in the multipotent stem cell niche in the central region of the body column. Ectoderm of the diploblastic hydra was shown to express higher levels of XPF as compared to the endoderm by semi-quantitative RT-PCR. Semi-quantitative RT-PCR analysis also demonstrated that interstitial cells, a multipotent and rapidly cycling stem cell lineage of hydra, express higher levels of XPF mRNA than other cell types. Our data show that XPF and by extension, the NER pathway is highly conserved during evolution. The prominent expression of an NER gene in interstitial cells may have implications for the lack of senescence in hydra. PMID:23577191
Coordinated regulation of XPA stability by ATR and HERC2 during nucleotide excision repair.
Lee, T-H; Park, J-M; Leem, S-H; Kang, T-H
ATR (ATM and Rad3-related) is an essential regulator of the nucleotide excision repair (NER) mechanism. For NER activation, ATR phosphorylates XPA, the rate-limiting factor in the NER pathway. However, the role of XPA phosphorylation at serine 196 by ATR has been elusive. Here we show that ATR-mediated XPA phosphorylation enhances XPA stability by inhibiting HERC2-mediated ubiquitination and subsequent degradation. We analyzed stabilization of XPA with substitutions of Ser 196 either to aspartate (S196D), a phosphomimetic mutation, or to alanine (S196A), a phosphodeficient mutation. Upon ultraviolet damage, ATR facilitated HERC2 dissociation from the XPA complex to induce XPA stabilization. However, this regulation was abrogated in S196A-complemented XPA-deficient cells due to persistent association of HERC2 with this XPA complex, resulting in enhanced ubiquitination of S196A. Conversely, the S196D substitution showed delayed degradation kinetics compared with the wild-type and less binding with HERC2, resulting in reduced ubiquitination of S196D. We also found that XPA phosphorylation enhanced the chromatin retention of XPA, the interaction with its binding partners following DNA damage. Taken together, our study presents a novel control mechanism in the NER pathway by regulating the steady-state level of XPA through posttranslational modifications by which ATR-mediated phosphorylation induces XPA stabilization by antagonizing HERC2-catalyzed XPA ubiquitination. PMID:23178497
Resistance to Nucleotide Excision Repair of Bulky Guanine Adducts Opposite Abasic Sites in DNA Duplexes and Relationships between Structure and Function
Liu, Zhi; Ding, Shuang; Kropachev, Konstantin; Lei, Jia; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E.
The nucleotide excision repair of certain bulky DNA lesions is abrogated in some specific non-canonical DNA base sequence contexts, while the removal of the same lesions by the nucleotide excision repair mechanism is efficient in duplexes in which all base pairs are complementary. Here we show that the nucleotide excision repair activity in human cell extracts is moderate-to-high in the case of two stereoisomeric DNA lesions derived from the pro-carcinogen benzo[a]pyrene (cis- and trans-B[a]P-N2-dG adducts) in a normal DNA duplex. By contrast, the nucleotide excision repair activity is completely abrogated when the canonical cytosine base opposite the B[a]P-dG adducts is replaced by an abasic site in duplex DNA. However, base excision repair of the abasic site persists. In order to understand the structural origins of these striking phenomena, we used NMR and molecular spectroscopy techniques to evaluate the conformational features of 11mer DNA duplexes containing these B[a]P-dG lesions opposite abasic sites. Our results show that in these duplexes containing the clustered lesions, both B[a]P-dG adducts adopt base-displaced intercalated conformations, with the B[a]P aromatic rings intercalated into the DNA helix. To explain the persistence of base excision repair in the face of the opposed bulky B[a]P ring system, molecular modeling results suggest how the APE1 base excision repair endonuclease, that excises abasic lesions, can bind productively even with the trans-B[a]P-dG positioned opposite the abasic site. We hypothesize that the nucleotide excision repair resistance is fostered by local B[a]P residue—DNA base stacking interactions at the abasic sites, that are facilitated by the absence of the cytosine partner base in the complementary strand. More broadly, this study sets the stage for elucidating the interplay between base excision and nucleotide excision repair in processing different types of clustered DNA lesions that are substrates of nucleotide
The endoperoxide ascaridol shows strong differential cytotoxicity in nucleotide excision repair-deficient cells
Abbasi, Rashda; Efferth, Thomas; Kuhmann, Christine; Opatz, Till; Hao, Xiaojiang; Popanda, Odilia; Schmezer, Peter
Targeting synthetic lethality in DNA repair pathways has become a promising anti-cancer strategy. However little is known about such interactions with regard to the nucleotide excision repair (NER) pathway. Therefore, cell lines with a defect in the NER genes ERCC6 or XPC and their normal counterparts were screened with 53 chemically defined phytochemicals isolated from plants used in traditional Chinese medicine for differential cytotoxic effects. The screening revealed 12 drugs that killed NER-deficient cells more efficiently than proficient cells. Five drugs were further analyzed for IC{sub 50} values, effects on cell cycle distribution, and induction of DNA damage. Ascaridol was the most effective compound with a difference of > 1000-fold in resistance between normal and NER-deficient cells (IC{sub 50} values for cells with deficiency in ERCC6: 0.15 μM, XPC: 0.18 μM, and normal cells: > 180 μM). NER-deficiency combined with ascaridol treatment led to G2/M-phase arrest, an increased percentage of subG1 cells, and a substantially higher DNA damage induction. These results were confirmed in a second set of NER-deficient and -proficient cell lines with isogenic background. Finally, ascaridol was characterized for its ability to generate oxidative DNA damage. The drug led to a dose-dependent increase in intracellular levels of reactive oxygen species at cytotoxic concentrations, but only NER-deficient cells showed a strongly induced amount of 8-oxodG sites. In summary, ascaridol is a cytotoxic and DNA-damaging compound which generates intracellular reactive oxidative intermediates and which selectively affects NER-deficient cells. This could provide a new therapeutic option to treat cancer cells with mutations in NER genes. -- Highlights: ► Thousand-fold higher Ascaridol activity in NER-deficient versus proficient cells. ► Impaired repair of Ascaridol-induced oxidative DNA damage in NER-deficient cells. ► Selective activity of Ascaridol opens new therapy
Role of base excision repair in maintaining the genetic and epigenetic integrity of CpG sites
Bellacosa, Alfonso; Drohat, Alexander C.
Cytosine methylation at CpG dinucleotides is a central component of epigenetic regulation in vertebrates, and the base excision repair (BER) pathway is important for maintaining both the genetic stability and the methylation status of CpG sites. This perspective focuses on two enzymes that are of particular importance for the genetic and epigenetic integrity of CpG sites, Methyl Binding Domain 4 (MBD4) and Thymine DNA Glycosylase (TDG). We discuss their capacity for countering C to T mutations at CpG sites, by initiating base excision repair of G·T mismatches generated by deamination of 5-methylcytosine (5mC). We also consider their role in active DNA demethylation, including pathways that are initiated by oxidation and/or deamination of 5mC. PMID:26021671
Nucleotide excision repair polymorphisms may modify ionizing radiation-related breast cancer risk in US radiologic technologists.
Rajaraman, Preetha; Bhatti, Parveen; Doody, Michele Morin; Simon, Steven L; Weinstock, Robert M; Linet, Martha S; Rosenstein, Marvin; Stovall, Marilyn; Alexander, Bruce H; Preston, Dale L; Sigurdson, Alice J
Exposure to ionizing radiation has been consistently associated with increased risk of female breast cancer. Although the majority of DNA damage caused by ionizing radiation is corrected by the base-excision repair pathway, certain types of multiple-base damage can only be repaired through the nucleotide excision repair pathway. In a nested case-control study of breast cancer in US radiologic technologists exposed to low levels of ionizing radiation (858 cases, 1,083 controls), we examined whether risk of breast cancer conferred by radiation was modified by nucleotide excision gene polymorphisms ERCC2 (XPD) rs13181, ERCC4 (XPF) rs1800067 and rs1800124, ERCC5 (XPG) rs1047769 and rs17655; and ERCC6 rs2228526. Of the 6 ERCC variants examined, only ERCC5 rs17655 showed a borderline main effect association with breast cancer risk (OR(GC) = 1.1, OR(CC) = 1.3; p-trend = 0.08), with some indication that individuals carrying the C allele variant were more susceptible to the effects of occupational radiation (EOR/Gy(GG) = 1.0, 95% CI = <0, 6.0; EOR/Gy(GC/CC) = 5.9, 95% CI = 0.9, 14.4; p(het) = 0.10). ERCC2 rs13181, although not associated with breast cancer risk overall, statistically significantly modified the effect of occupational radiation dose on risk of breast cancer (EOR/Gy(AA) = 9.1, 95% CI = 2.1-21.3; EOR/Gy(AC/CC) = 0.6, 95% CI = <0, 4.6; p(het) = 0.01). These results suggest that common variants in nucleotide excision repair genes may modify the association between occupational radiation exposure and breast cancer risk. PMID:18767034
Evidence for an involvement of thymidine kinase in the excision repair of ultraviolet-irradiated herpes simplex virus in human cells
Intine, R.V.; Rainbow, A.J. )
A wild-type strain of herpes simplex virus type 1 (HSV-1:KOS) encoding a functional thymidine kinase (tk+) and a tk- mutant strain (HSV-1:PTK3B) were used to study the role of the viral tk in the repair of UV-irradiated HSV-1 in human cells. UV survival of HSV-1:PTK3B was substantially reduced compared with that of HSV-1:KOS when infecting normal human cells. In contrast, the UV survival of HSV-1:PTK3B was similar to that of HSV-1:KOS when infecting excision repair-deficient cells from a xeroderma pigmentosum patient from complementation group A. These results suggest that the repair of UV-irradiated HSV-1 in human cells depends, in part at least, on expression of the viral tk and that the repair process influenced by tk activity is excision repair or a process dependent on excision repair.
Repair of rDNA in Saccharomyces cerevisiae: RAD4-independent strand-specific nucleotide excision repair of RNA polymerase I transcribed genes.
Verhage, R A; Van de Putte, P; Brouwer, J
Removal of UV-induced pyrimidine dimers from the individual strands of the rDNA locus in Saccharomyces cerevisiae was studied. Yeast rDNA, that is transcribed by RNA polymerase I(RNA pol I), is repaired efficiently, slightly strand-specific and independently of RAD26, which has been implicated in transcription-coupled repair of the RNA pol II transcribed RPB2 gene. No repair of rDNA is observed in rad1,2,3 and 14 mutants, demonstrating that dimer removal from this highly repetitive DNA is accomplished by nucleotide excision repair (NER). In rad7 and rad16 mutants, which are specifically deficient in repair of non-transcribed DNA, there is a clear preferential repair of the transcribed strand of rDNA, indicating that strand-specific and therefore probably transcription-coupled repair of RNA pol I transcribed genes does exist in yeast. Unexpectedly, the transcribed but not the non-transcribed strand of rDNA can be repaired in rad4 mutants, which seem otherwise completely NER-deficient. PMID:8604332
Metal inhibition of human alkylpurine-DNA-N-glycosylase activityin base excision repair
Wang, Ping; Guliaev, Anton B.; Hang, Bo
Cadmium (Cd{sup 2+}), nickel (Ni{sup 2+}) and cobalt (Co{sup 2+}) are human and/or animal carcinogens. Zinc (Zn{sup 2+}) is not categorized as a carcinogen, and rather an essential element to humans. Metals were recently shown to inhibit DNA repair proteins that use metals for their function and/or structure. Here we report that the divalent ions Cd{sup 2+}, Ni{sup 2+}, and Zn{sup 2+} can inhibit the activity of a recombinant human N-methylpurine-DNA glycosylase (MPG) toward a deoxyoligonucleotide with ethenoadenine (var epsilonA). MPG removes a variety of toxic/mutagenic alkylated bases and does not require metal for its catalytic activity or structural integrity. At concentrations starting from 50 to 1000 {micro}M, both Cd{sup 2+} and Zn{sup 2+} showed metal-dependent inhibition of the MPG catalytic activity. Ni{sup 2+} also inhibited MPG, but to a lesser extent. Such an effect can be reversed with EDTA addition. In contrast, Co{sup 2+} and Mg{sup 2+} did not inhibit the MPG activity in the same dose range. Experiments using HeLa cell-free extracts demonstrated similar patterns of inactivation of the var epsilonA excision activity by the same metals. Binding of MPG to the substrate was not significantly affected by Cd{sup 2+}, Zn{sup 2+}, and Ni{sup 2+} at concentrations that show strong inhibition of the catalytic function, suggesting that the reduced catalytic activity is not due to altered MPG binding affinity to the substrate. Molecular dynamics (MD) simulations with Zn{sup 2+} showed that the MPG active site has a potential binding site for Zn{sup 2+}, formed by several catalytically important and conserved residues. Metal binding to such a site is expected to interfere with the catalytic mechanism of this protein. These data suggest that inhibition of MPG activity may contribute to metal genotoxicity and depressed repair of alkylation damage by metals in vivo.
XRCC1 and Base Excision Repair Balance in Response to Nitric Oxide
Mutamba, James T.; Svilar, David; Prasongtanakij, Somsak; Wang, Xiao-Hong; Lin, Ying-Chih; Dedon, Peter C.; Sobol, Robert W.; Engelward, Bevin P.
Inflammation associated reactive oxygen and nitrogen species (RONs), including peroxynitrite (ONOO−) and nitric oxide (NO· ), create base lesions that potentially play a role in the toxicity and large-scale genomic rearrangements associated with many malignancies. Nevertheless, little is known about the functional role of base excision repair (BER) deficiencies following exposure to RONs. Here, we explore the role of XRCC1 in modulating the levels of RONs-induced genotoxicity. XRCC1 is a scaffold protein critical for BER for which polymorphisms modulate the risk of cancer. We exploited CHO and human glioblastoma cell lines engineered to carry varied levels of BER components to study XRCC1. Cytotoxicity and SSB-intermediate levels were evaluated following exposure to the ONOO− donor, SIN-1, and to gaseous NO·. XRCC1 null cells are slightly more sensitive to SIN-1 toxicity. To explore the potential importance of XRCC1 in response to NO· -induced lesions, we used small-scale bioreactors to expose cells to NO· and found that XRCC1 does not impact genotoxicity in CHO cells, suggesting a minimal role for XRCC1 in response to RONs. However, using a molecular beacon assay to measure AAG-mediated lesion removal in vitro, we found that XRCC1 facilitates AAG-initiated BER of two key NO· -induced lesions: 1,N6-ethenoadenine and hypoxanthine. Furthermore, overexpression of AAG rendered XRCC1 cells sensitive to NO· -induced DNA damage and toxicity. These results show that AAG is a key glycosylase in response to NO· exposure and that the cellular and functional impact of XRCC1 depends upon the level of AAG. These studies are some of the first to assess the functional role of XRCC1 in response to NO·, and demonstrate the importance of BER balance when considering the impact of XRCC1 polymorphisms in response to RONs. PMID:22041025
Variation in DNA Base Excision Repair Genes in Fuchs Endothelial Corneal Dystrophy
Wójcik, Katarzyna A.; Synowiec, Ewelina; Polakowski, Piotr; Błasiak, Janusz; Szaflik, Jerzy; Szaflik, Jacek P.
Background Fuchs endothelial corneal dystrophy (FECD) is a corneal disease characterized by abnormalities in the Descemet membrane and the corneal endothelium. The etiology of this disease is poorly understood. An increased level of oxidative DNA damage reported in FECD corneas suggests a role of DNA base excision repair (BER) genes in its pathogenesis. In this work, we searched for the association between variation of the PARP-1, NEIL1, POLG, and XRCC1 genes and FECD occurrence. Material/Methods This study was conducted on 250 FECD patients and 353 controls using polymerase chain reaction-restriction fragment length polymorphism, high-resolution melting analysis, and the TaqMan® SNP Genotyping Assay. Results We observed that the A/A genotype and the A allele of the c.1196A>G polymorphism of the XRCC1 gene were positively correlated with an increased FECD occurrence, whereas the G allele had the opposite effect. A weak association between the C/G genotype of the g.46438521G>C polymorphism of the NEIL1 gene and an increased incidence of FECD was also detected. Haplotypes of both polymorphisms of the XRCC1 were associated with FECD occurrence. No association of the c.2285T>C, c.–1370T>A and c.580C>T polymorphisms of the PARP-1, POLG and XRCC1 genes, respectively, with FECD occurrence was observed. Conclusions Our results suggest that the c.1196A>G polymorphism in the XRCC1 gene may be an independent genetic risk factor for FECD. PMID:26388025
Germ line variation in nucleotide excision repair genes and lung cancer risk in smokers.
Sakoda, Lori C; Loomis, Melissa M; Doherty, Jennifer A; Julianto, Liberto; Barnett, Matt J; Neuhouser, Marian L; Thornquist, Mark D; Weiss, Noel S; Goodman, Gary E; Chen, Chu
Since nucleotide excision repair (NER) is primarily responsible for detecting and removing bulky DNA lesions induced by tobacco smoke in the respiratory tract, single nucleotide polymorphisms (SNPs) in NER protein-encoding genes may influence lung cancer risk, particularly in smokers. Studies testing this hypothesis have produced inconsistent results, with most analyzing a few SNPs in relatively small population samples. In a study nested in the Beta- Carotene and Retinol Efficacy Trial, we examined 79 tag and previously reported risk-associated SNPs in the ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, LIG1, POLE, XPA, and XPC genes in 744 lung cancer cases and 1,477 controls, all of whom were non-Hispanic white smokers. Using logistic regression, odds ratios (OR) and 95% confidence intervals (95% CI) were calculated to estimate lung cancer risk associated with SNP genotypes and haplotypes, adjusting for case-control matching factors. Lung cancer risk was modestly associated with LIG1 rs156640 (OR per G allele, 1.23; 95% CI, 1.08-1.40), rs156641 (OR per A allele, 1.23; 95% CI, 1.08-1.40), and rs8100261 (OR per A allele, 0.83; 95% CI, 0.76-0.98); XPA rs3176658 (OR per A allele, 0.83; 95% CI, 0.69-1.00); and ERCC2 rs50871 (OR per C allele, 1.15; 95% CI: 1.01-1.30). Associations with LIG1 and XPA, but not ERCC2, haplotypes were found. The results of this study and others suggest that inherited variants in LIG1 and possibly other NER genes may predispose to smoking-related lung cancer. Given that chance likely accounts for one or more of the associations observed, replication of our findings is needed. PMID:22493747
Nucleotide excision repair DNA synthesis by excess DNA polymerase beta: a potential source of genetic instability in cancer cells.
Canitrot, Y; Hoffmann, J S; Calsou, P; Hayakawa, H; Salles, B; Cazaux, C
The nucleotide excision repair pathway contributes to genetic stability by removing a wide range of DNA damage through an error-free reaction. When the lesion is located, the altered strand is incised on both sides of the lesion and a damaged oligonucleotide excised. A repair patch is then synthesized and the repaired strand is ligated. It is assumed that only DNA polymerases delta and/or epsilon participate to the repair DNA synthesis step. Using UV and cisplatin-modified DNA templates, we measured in vitro that extracts from cells overexpressing the error-prone DNA polymerase beta exhibited a five- to sixfold increase of the ultimate DNA synthesis activity compared with control extracts and demonstrated the specific involvement of Pol beta in this step. By using a 28 nt gapped, double-stranded DNA substrate mimicking the product of the incision step, we showed that Pol beta is able to catalyze strand displacement downstream of the gap. We discuss these data within the scope of a hypothesis previously presented proposing that excess error-prone Pol beta in cancer cells could perturb the well-defined specific functions of DNA polymerases during error-free DNA transactions. PMID:10973926
NDR1 modulates the UV-induced DNA-damage checkpoint and nucleotide excision repair
Park, Jeong-Min; Choi, Ji Ye; Yi, Joo Mi; Chung, Jin Woong; Leem, Sun-Hee; Koh, Sang Seok; Kang, Tae-Hong
Nucleotide excision repair (NER) is the sole mechanism of UV-induced DNA lesion repair in mammals. A single round of NER requires multiple components including seven core NER factors, xeroderma pigmentosum A–G (XPA–XPG), and many auxiliary effector proteins including ATR serine/threonine kinase. The XPA protein helps to verify DNA damage and thus plays a rate-limiting role in NER. Hence, the regulation of XPA is important for the entire NER kinetic. We found that NDR1, a novel XPA-interacting protein, modulates NER by modulating the UV-induced DNA-damage checkpoint. In quiescent cells, NDR1 localized mainly in the cytoplasm. After UV irradiation, NDR1 accumulated in the nucleus. The siRNA knockdown of NDR1 delayed the repair of UV-induced cyclobutane pyrimidine dimers in both normal cells and cancer cells. It did not, however, alter the expression levels or the chromatin association levels of the core NER factors following UV irradiation. Instead, the NDR1-depleted cells displayed reduced activity of ATR for some set of its substrates including CHK1 and p53, suggesting that NDR1 modulates NER indirectly via the ATR pathway. - Highlights: • NDR1 is a novel XPA-interacting protein. • NDR1 accumulates in the nucleus in response to UV irradiation. • NDR1 modulates NER (nucleotide excision repair) by modulating the UV-induced DNA-damage checkpoint response.
DNA polymerase beta-catalyzed-PCNA independent long patch base excision repair synthesis: a mechanism for repair of oxidatively damaged DNA ends in post-mitotic brain.
Wei, Wei; Englander, Ella W
Oxidative DNA damage incidental to normal respiratory metabolism poses a particular threat to genomes of highly metabolic-long lived cells. We show that post-mitotic brain has capacity to repair oxidatively damaged DNA ends, which are targets of the long patch (LP) base excision repair (BER) subpathway. LP-BER relies, in part, on proteins associated with DNA replication, including proliferating cell nuclear antigen and is inherent to proliferating cells. Nonetheless, repair products are generated with brain extracts, albeit at slow rates, in the case of 5'-DNA ends modeled with tetrahydrofuran (THF). THF at this position is refractory to DNA polymerase beta 5'-deoxyribose 5-phosphate lyase activity and drives repair into the LP-BER subpathway. Comparison of repair of 5'-THF-blocked termini in the post-mitotic rat brain and proliferative intestinal mucosa, revealed that in mucosa, resolution of damaged 5'-termini is accompanied by formation of larger repair products. In contrast, adducts targeted by the single nucleotide BER are proficiently repaired with both extracts. Our findings reveal mechanistic differences in BER processes selective for the brain versus proliferative tissues. The differences highlight the physiological relevance of the recently proposed 'Hit and Run' mechanism of alternating cleavage/synthesis steps, in the proliferating cell nuclear antigen-independent LP-BER process. PMID:18752643
Involvement of mammalian OGG1(MMH) in excision of the 8-hydroxyguanine residue in DNA.
Nishimura, Susumu
8-Hydroxyguanine (7,8-dihydro-8-oxoguanine, abbreviated as 8-OH-G or 8-oxoG) is the site of a frequent mutagenic DNA lesion produced by oxidative damage. MutM of E. coli and OGG1 of Saccharomyces cervisiae are known to possess 8-OH-G glycosylase and apurinic (AP) site lyase activity. cDNA clones of four isoforms (types 1a, 1b, 1c, and 2) of human OGG1 homologs (hMMH) were isolated. In order to examine whether expression of hMMH (hOGG1) protein actually occurs in human cells, we prepared type 1a specific antibody, and by using this antibody, we showed that type 1a protein isolated from HeLaS3 has 8-OH-G glycosylase/lyase activity. Furthermore, we showed that type 1a protein is a major enzyme for repair of the 8-OH-G lesion in human cells. In our second study, we generated a mouse line carrying an inactivated mutant Mmh allele by targeted gene disruption. Liver extracts of Mmh homozygous mutant mice were found to have loss of the nicking activity for the 8-OH-G site. In addition, the amount of endogenous 8-OH-G in liver DNA of the homozygous mice increased linearly with age, reaching 7-fold increase in 14 week old mice, over that of wild-type or heterozygous mice. Furthermore, when homozygous mice were fed the oxygen radical-forming agent KBrO3, to provide oxidative stress, the level of 8-OH-G in kidney DNA was tremendously increased: more than 200-fold as that of control mice without oxidative stress after 12 weeks of age. These results indicate that Ogg1/Mmh plays an essential role in the repair of the 8-OH-G residue in DNA produced by oxidative stress. PMID:11978483
In situ analysis of repair processes for oxidative DNA damage in mammalian cells
NASA Astrophysics Data System (ADS)
Lan, Li; Nakajima, Satoshi; Oohata, Yoshitsugu; Takao, Masashi; Okano, Satoshi; Masutani, Mitsuko; Wilson, Samuel H.; Yasui, Akira
Oxidative DNA damage causes blocks and errors in transcription and replication, leading to cell death and genomic instability. Although repair mechanisms of the damage have been extensively analyzed in vitro, the actual in vivo repair processes remain largely unknown. Here, by irradiation with an UVA laser through a microscope lens, we have conditionally produced single-strand breaks and oxidative base damage at restricted nuclear regions of mammalian cells. We showed, in real time after irradiation by using antibodies and GFP-tagged proteins, rapid and ordered DNA repair processes of oxidative DNA damage in human cells. Furthermore, we characterized repair pathways by using repair-defective mammalian cells and found that DNA polymerase accumulated at single-strand breaks and oxidative base damage by means of its 31- and 8-kDa domains, respectively, and that XRCC1 is essential for both polymerase -dependent and proliferating cell nuclear antigen-dependent repair pathways of single-strand breaks. Thus, the repair of oxidative DNA damage is based on temporal and functional interactions among various proteins operating at the site of DNA damage in living cells.
Rapid and apparently error-prone excision repair of nonreplicating UV-irradiated plasmids in Xenopus laevis oocytes
Hays, J.B.; Ackerman, E.J.; Pang, Q.S. )
Repair of UV-irradiated plasmid DNA microinjected into frog oocytes was measured by two techniques: transformation of repair-deficient (delta uvrB delta recA delta phr) bacteria, and removal of UV endonuclease-sensitive sites (ESS). Transformation efficiencies relative to unirradiated plasmids were used to estimate the number of lethal lesions; the latter were assumed to be Poisson distributed. These estimates were in good agreement with measurements of ESS. By both criteria, plasmid DNA was efficiently repaired, mostly during the first 2 h, when as many as 2 x 10(10) lethal lesions were removed per oocyte. This rate is about 10(6) times the average for removal of ESS from repair-proficient human cells. Repair was slower but still significant after 2 h, but some lethal lesions usually remained after overnight incubation. Most repair occurred in the absence of light, in marked contrast to differentiated frog cells, previously shown to possess photoreactivating but no excision repair activity. There was no increase in the resistance to DpnI restriction of plasmids (methylated in Escherichia coli at GATC sites) incubated in oocytes; this implies no increase in hemimethylated GATC sites, and hence no semiconservative DNA replication. Plasmid substrates capable of either intramolecular or intermolecular homologous recombination were not recombined, whether UV-irradiated or not. Repair of Lac+ plasmids was accompanied by a significant UV-dependent increase in the frequency of Lac- mutants, corresponding to a repair synthesis error frequency on the order of 10(-4) per nucleotide.
The nucleotide excision repair system of Borrelia burgdorferi is the sole pathway involved in repair of DNA damage by UV light.
Hardy, Pierre-Olivier; Chaconas, George
To survive and avoid accumulation of mutations caused by DNA damage, the genomes of prokaryotes encode a variety of DNA repair pathways most well characterized in Escherichia coli. Some of these are required for the infectivity of various pathogens. In this study, the importance of 25 DNA repair/recombination genes for Borrelia burgdorferi survival to UV-induced DNA damage was assessed. In contrast to E. coli, where 15 of these genes have an effect on survival of UV irradiation, disruption of recombinational repair, transcription-coupled repair, methyl-directed mismatch correction, and repair of arrested replication fork pathways did not decrease survival of B. burgdorferi exposed to UV light. However, the disruption of the B. burgdorferi nucleotide excision repair (NER) pathway (uvrA, uvrB, uvrC, and uvrD) resulted in a 10- to 1,000-fold increase in sensitivity to UV light. A functional NER pathway was also shown to be required for B. burgdorferi resistance to nitrosative damage. Finally, disruption of uvrA, uvrC, and uvrD had only a minor effect upon murine infection by increasing the time required for dissemination. PMID:23475971
Silibinin enhances the repair of ultraviolet B-induced DNA damage by activating p53-dependent nucleotide excision repair mechanism in human dermal fibroblasts.
Guillermo-Lagae, Ruth; Deep, Gagan; Ting, Harold; Agarwal, Chapla; Agarwal, Rajesh
Ultraviolet radiation B (UVB) is the main cause of DNA damage in epidermal cells; and if not repaired, this DNA damage leads to skin cancer. In earlier studies, we have reported that natural flavonolignan silibinin exerts strong chemopreventive efficacy against UVB-induced skin damage and carcinogenesis; however mechanistic studies are still being actively pursued. Here, we investigated the role of nucleotide excision repair (NER) pathway in silibinin's efficacy to repair UVB-induced DNA damage. Normal human dermal fibroblasts (NHDFs) were exposed to UVB (1 mJ/cm2) with pre- or post- silibinin (100 μM) treatment, and cyclobutane pyrimidine dimers (CPDs) formation/repair was measured. Results showed that post-UVB silibinin treatment accelerates DNA repair via activating the NER pathway including the expression of XPA (xeroderma pigmentosum complementation group A), XPB, XPC, and XPG. In UVB exposed fibroblasts, silibinin treatment also increased p53 and GADD45α expression; the key regulators of the NER pathway and DNA repair. Consistently, post-UVB silibinin treatment increased the mRNA transcripts of XPA and GADD45α. Importantly, silibinin showed no effect on UVB-induced DNA damage repair in XPA- and XPB-deficient human dermal fibroblasts suggesting their key role in silibinin-mediated DNA damage repair. Moreover, in the presence of pifithrin-α, an inhibitor of p53, the DNA repair efficacy of silibinin was compromised associated with a reduction in XPA and GADD45α transcripts. Together, these findings suggest that silibinin's efficacy against UVB-induced photodamage is primarily by inhibiting NER and p53; and these findings further support silibinin's usage as a potential inexpensive, effective, and non-toxic agent for skin cancer chemoprevention. PMID:26447614
Base excision repair of both uracil and oxidatively damaged bases contribute to thymidine deprivation-induced radiosensitization
Allen, Bryan G.; Johnson, Monika; Marsh, Anne E.; Dornfeld, Kenneth J. . E-mail: email@example.com
Purpose: Increased cellular sensitivity to ionizing radiation due to thymidine depletion is the basis of radiosensitization with fluoropyrimidine and methotrexate. The mechanism responsible for cytotoxicity has not been fully elucidated but appears to involve both the introduction of uracil into, and its removal from, DNA. The role of base excision repair of uracil and oxidatively damaged bases in creating the increased radiosensitization during thymidine depletion is examined. Methods and Materials: Isogenic strains of S. cerevisiae differing only at loci involved in DNA repair functions were exposed to aminopterin and sulfanilamide to induce thymidine deprivation. Cultures were irradiated and survival determined by clonogenic survival assay. Results: Strains lacking uracil base excision repair (BER) activities demonstrated less radiosensitization than the parental strain. Mutant strains continued to show partial radiosensitization with aminopterin treatment. Mutants deficient in BER of both uracil and oxidatively damaged bases did not demonstrate radiosensitization. A recombination deficient rad52 mutant strain was markedly sensitive to radiation; addition of aminopterin increased radiosensitivity only slightly. Radiosensitization observed in rad52 mutants was also abolished by deletion of the APN1, NTG1, and NTG2 genes. Conclusion: These data suggest radiosensitization during thymidine depletion is the result of BER activities directed at both uracil and oxidatively damaged bases.
A unified view of base excision repair: lesion-dependent protein complexes regulated by post-translational modification
Almeida, Karen H.; Sobol, Robert W.
Base excision repair (BER) proteins act upon a significantly broad spectrum of DNA lesions that result from endogenous and exogenous sources. Multiple sub-pathways of BER (short-path or long-patch) and newly designated DNA repair pathways (e.g., SSBR and NIR) that utilize BER proteins complicate any comprehensive understanding of BER and its role in genome maintenance, chemotherapeutic response, neurodegeneration, cancer or aging. Herein, we propose a unified model of BER, comprised of three functional processes: Lesion Recognition/Strand Scission, Gap Tailoring and DNA Synthesis/Ligation, each represented by one or more multiprotein complexes and coordinated via the XRCC1/DNA Ligase III and PARP1 scaffold proteins. BER therefore may be represented by a series of repair complexes that assemble at the site of the DNA lesion and mediates repair in a coordinated fashion involving protein-protein interactions that dictate subsequent steps or sub-pathway choice. Complex formation is influenced by post-translational protein modifications that arise from the cellular state or the DNA damage response, providing an increase in specificity and efficiency to the BER pathway. In this review, we have summarized the reported BER protein-protein interactions and protein post-translational modifications and discuss the impact on DNA repair capacity and complex formation. PMID:17337257
Initiation of DNA Interstrand Cross-link Repair in Mammalian Cells
Hlavin, Erica M.; Smeaton, Michael B.; Miller, Paul S.
Interstrand cross-links (ICLs) are among the most cytotoxic DNA lesions to cells because they prevent the two DNA strands from separating, thereby precluding replication and transcription. Even though chemotherapeutic cross-linking agents are well established in clinical use, and numerous repair proteins have been implicated in the initial events of mammalian ICL repair, the precise mechanistic details of these events remain to be elucidated. This review will summarize our current understanding of how ICL repair is initiated with an emphasis on the context (replicating, transcribed or quiescent DNA) in which the ICL is recognized, and how the chemical and physical properties of ICLs influence repair. Although most studies have focused on replication-dependent repair because of the relation to highly replicative tumor cells, replication-independent ICL repair is likely to be important in the circumvention of cross-link cytotoxicity in non-dividing, terminally differentiated cells that may be challenged with exogenous or endogenous sources of ICLs. Consequently, the ICL repair pathway that should be considered 'dominant' appears to depend on the cell type and the DNA context in which the ICL is encountered. The ability to define and inhibit distinct pathways of ICL repair in different cell cycle phases may help in developing methods that increase cytotoxicity to cancer cells while reducing side-effects in non-dividing normal cells. This may also lead to a better understanding of pathways that protect against malignancy and aging. PMID:20658650
Elevated metals compromise repair of oxidative DNA damage via the base excision repair pathway: implications of pathologic iron overload in the brain on integrity of neuronal DNA.
Li, Hui; Swiercz, Rafal; Englander, Ella W
Tissue-specific iron content is tightly regulated to simultaneously satisfy specialized metabolic needs and avoid cytotoxicity. In the brain, disruption of iron homeostasis may occur in acute as well as progressive injuries associated with neuronal dysfunction and death. We hypothesized that adverse effects of disrupted metal homeostasis on brain function may involve impairment of DNA repair processes. Because in the brain, the base excision repair (BER) pathway is central for handling oxidatively damaged DNA, we investigated effects of elevated iron and zinc on key BER enzymes. In vitro DNA repair assays revealed inhibitory effects of metals on BER activities, including the incision of abasic sites, 5'-flap cleavage, gap filling DNA synthesis and ligation. Using the comet assay, we showed that while metals at concentrations which inhibit BER activities in in vitro assays, did not induce direct genomic damage in cultured primary neurons, they significantly delayed repair of genomic DNA damage induced by sublethal exposure to H(2)O(2). Thus, in the brain even a mild transient metal overload, may adversely affect the DNA repair capacity and thereby compromise genomic integrity and initiate long-term deleterious sequelae including neuronal dysfunction and death. PMID:19619136
Telomere proteins POT1, TRF1 and TRF2 augment long-patch base excision repair in vitro
Miller, Adam S; Balakrishnan, Lata; Buncher, Noah A
Human telomeres consist of multiple tandem hexameric repeats, each containing a guanine triplet. Guanosine-rich clusters are highly susceptible to oxidative base damage, necessitating base excision repair (BER). Previous demonstration of enhanced strand displacement synthesis by the BER component DNA polymerase β in the presence of telomere protein TRF2 suggests that telomeres employ long-patch (LP) BER. Earlier analyses in vitro showed that efficiency of BER reactions is reduced in the DNA-histone environment of chromatin. Evidence presented here indicates that BER is promoted at telomeres. We found that the three proteins that contact telomere DNA, POT1, TRF1 and TRF2, enhance the rate of individual steps of LP-BER and stimulate the complete reconstituted LP-BER pathway. Thought to protect telomere DNA from degradation, these proteins still apparently evolved to allow selective access of repair proteins. PMID:22336916
MUTYH-associated polyposis (MAP), the syndrome implicating base excision repair in inherited predisposition to colorectal tumors
Venesio, Tiziana; Balsamo, Antonella; D'Agostino, Vito G.; Ranzani, Guglielmina N.
In 2002, Al-Tassan and co-workers described for the first time a recessive form of inherited polyposis associated with germline mutations of MUTYH, a gene encoding a base excision repair (BER) protein that counteracts the DNA damage induced by the oxidative stress. MUTYH-associated polyposis (MAP) is now a well-defined cancer susceptibility syndrome, showing peculiar molecular features that characterize disease progression. However, some aspects of MAP, including diagnostic criteria, genotype-phenotype correlations, pathogenicity of variants, as well as relationships between BER and other DNA repair pathways, are still poorly understood. A deeper knowledge of the MUTYH expression pattern is likely to refine our understanding of the protein role and, finally, to improve guidances for identifying and handling MAP patients. PMID:22876359
Inhibition of DNA excision repair by methotrexate in Chinese hamster ovary cells following exposure to ultraviolet irradiation or ethylmethanesulfonate
Borchers, A.H.; Kennedy, K.A.; Straw, J.A. )
Previous results have suggested that methotrexate (MTX) could interfere with the repair of spontaneous DNA damage. To determine its effects on induced DNA damage, MTX was compared to hydroxyurea and arabinofuranosylcytosine (H/A), a drug combination known to block the DNA polymerase step of excision repair, for its ability to cause the accumulation of single-strand breaks (SSB) following exposure to either UV light or the alkylating agent ethylmethanesulfonate in Chinese hamster ovary cells. SSB were measured by alkaline elution 1, 2, and 6 h after exposure to either 1.8 mg/ml of ethylmethanesulfonate or 10 J/m2 of UV in cells pretreated with MTX or H/A. Following exposure to ethylmethanesulfonate, significant accumulation of SSB occurred in cells pretreated with either H/A or MTX. Coadministration of hypoxanthine and thymidine in MTX-treated cells prevented SSB accumulation, indicating that nucleotide depletion by MTX had inhibited repair synthesis. After UV irradiation, SSB accumulation was much less in MTX- than in H/A-treated cells. MTX was found to have no effect on the incision of UV damage. These results indicate that nucleotide depletion by MTX can affect the repair of DNA damage by exogenous agents, and that the extent of inhibition is dependent on the type of damage induced.
Circadian control of XPA and excision repair of cisplatin-DNA damage by cryptochrome and HERC2 ubiquitin ligase.
Kang, Tae-Hong; Lindsey-Boltz, Laura A; Reardon, Joyce T; Sancar, Aziz
Cisplatin is one of the most commonly used anticancer drugs. It kills cancer cells by damaging their DNA, and hence cellular DNA repair capacity is an important determinant of its efficacy. Here, we investigated the repair of cisplatin-induced DNA damage in mouse liver and testis tissue extracts prepared at regular intervals over the course of a day. We find that the XPA protein, which plays an essential role in repair of cisplatin damage by nucleotide excision repair, exhibits circadian oscillation in the liver but not in testis. Consequently, removal of cisplatin adducts in liver extracts, but not in testis extracts, exhibits a circadian pattern with zenith at approximately 5 pm and nadir at approximately 5 am. Furthermore, we find that the circadian oscillation of XPA is achieved both by regulation of transcription by the core circadian clock proteins including cryptochrome and by regulation at the posttranslational level by the HERC2 ubiquitin ligase. These findings may be used as a guide for timing of cisplatin chemotherapy. PMID:20304803
Functional interactions and signaling properties of mammalian DNA mismatch repair proteins.
Bellacosa, A
The mismatch repair (MMR) system promotes genomic fidelity by repairing base-base mismatches, insertion-deletion loops and heterologies generated during DNA replication and recombination. This function is critically dependent on the assembling of multimeric complexes involved in mismatch recognition and signal transduction to downstream repair events. In addition, MMR proteins coordinate a complex network of physical and functional interactions that mediate other DNA transactions, such as transcription-coupled repair, base excision repair and recombination. MMR proteins are also involved in activation of cell cycle checkpoint and induction of apoptosis when DNA damage overwhelms a critical threshold. For this reason, they play a role in cell death by alkylating agents and other chemotherapeutic drugs, including cisplatin. Inactivation of MMR genes in hereditary and sporadic cancer is associated with a mutator phenotype and inhibition of apoptosis. In the future, a deeper understanding of the molecular mechanisms and functional interactions of MMR proteins will lead to the development of more effective cancer prevention and treatment strategies. PMID:11687886
Enhanced excision repair and lack of PSII activity contribute to higher UV survival of Chlamydomonas reinhardtii cells in dark.
Chaudhari, Vishalsingh R; Vyawahare, Aniket; Bhattacharjee, Swapan K; Rao, Basuthkar J
Plant cells are known to differentiate their responses to stress depending up on the light conditions. We observed that UVC sensitive phenotype of light grown asynchronous Chlamydomonas reinhardtii culture (Light culture: LC) can be converted to relatively resistant form by transfer to dark condition (Dark culture: DC) before UVC exposure. The absence of photosystem II (PSII) function, by either atrazine treatment in wild type or in D1 (psbA) null mutant, conferred UV protection even in LC. We provide an indirect support for involvement of reactive oxygen species (ROS) signalling by showing higher UV survival on exposures to mild dose of H2O2 or Methyl Viologen. Circadian trained culture also showed a rhythmic variation in UV sensitivity in response to alternating light-dark (12 h:12 h) entrainment, with maximum UV survival at the end of 12 h dark and minimum at the end of 12 h light. This rhythm failed to maintain in "free running" conditions, making it a non-circadian phenotype. Moreover, atrazine strongly inhibited rhythmic UV sensitivity and conferred a constitutively high resistance, without affecting internal circadian rhythm marker expression. Dampening of UV sensitivity rhythm in Thymine-dimer excision repair mutant (cc-888) suggested the involvement of DNA repair in this phenomenon. DNA excision repair (ER) assays in cell-free extracts revealed that dark incubated cells exhibit higher ER compared to those growing in light, underscoring the role of ER in conferring differential UV sensitivity in dark versus light incubation. We suggest that multiple factors such as ROS changes triggered by differences in PSII activity, concomitant with differential ER efficiency collectively contribute to light-dark (12 h: 12 h) rhythmicity in C. reinhardtii UV sensitivity. PMID:25660990
A mutant of Eshcerchia coli K-12, URT-43, with a temperature-sensitive defect at the incision step of the excision repair mechanism.
Morimyo, M; Suzuki, K; Shimauzu, Y
URT-43, which has a defect in excision repair, exhibits a temperature-dependent ultraviolet survival. It was shown that URT-43 requires protein synthesis but not DNA synthesis for recovery, by examining recovery in a growth medium containing chloramphenicol or nalidixic acid. The recovery of irradiated bacteriophage lambda in URT-43 took place in a medium containing nalidixic acid at 30 degrees, but not at 41 degrees, and chloramphenicol prevented this recovery. These results seem to imply that the product of the mutated gene in URT-43 is labile. URT-43 was confirmed to have a temperature-sensitive mutation at the incision step of the excision repair mechanism by examining the nick formation of parental DNA in alkaline sucrose gradients. The release of pyrimidine dimers was reinvestigated directly by one- and two-dimensional paper-chromatography and indirectly by examining the distribution of DNA molecules synthesized after irradiation. Dimers were excised into the acid-soluble fraction when growing bacteria were incubated, but were not excised when in amino acid starved bacteria. These results suggest that URT-43 is a mutant slowly excising pyrimidine dimers because the product of a mutated gene concerned with the incision step of the excision repair mechanism is unstable. PMID:1093010
Active transcriptomic and proteomic reprogramming in the C. elegans nucleotide excision repair mutant xpa-1.
Kassahun, Henok; Nilsen, Hilde
Oxidative stress promotes human aging and contributes to common neurodegenerative diseases. Endogenous DNA damage induced by oxidative stress is believed to be an important promoter of neurodegenerative diseases. Although a large amount of evidence correlates a reduced DNA repair capacity with aging and neurodegenerative disease, there is little direct evidence of causality. Moreover, the contribution of oxidative DNA damage to the aging process is poorly understood. We have used the nematode Caenorhabditis elegans to study the contribution of oxidative DNA damage and repair to aging. C. elegans is particularly well suited to tackle this problem because it has a minimum complexity DNA repair system, which enables us to circumvent the important limitation presented by the extensive redundancy of DNA repair enzymes in mammals. PMID:24744987
XPC is essential for nucleotide excision repair of zidovudine-induced DNA damage in human hepatoma cells
Wu Qiangen; Beland, Frederick A.; Chang, Ching-Wei; Fang Jialong
Zidovudine (3'-azido-3'-dexoythymidine, AZT), a nucleoside reverse transcriptase inhibitor, can be incorporated into DNA and cause DNA damage. The mechanisms underlying the repair of AZT-induced DNA damage are unknown. To investigate the pathways involved in the recognition and repair of AZT-induced DNA damage, human hepatoma HepG2 cells were incubated with AZT for 2 weeks and the expression of DNA damage signaling pathways was determined using a pathway-based real-time PCR array. Compared to control cultures, damaged DNA binding and nucleotide excision repair (NER) pathways showed significantly increased gene expression. Further analysis indicated that AZT treatment increased the expression of genes associated with NER, including XPC, XPA, RPA1, GTF2H1, and ERCC1. Western blot analysis demonstrated that the protein levels of XPC and GTF2H1 were also significantly up-regulated. To explore further the function of XPC in the repair of AZT-induced DNA damage, XPC expression was stably knocked down by 71% using short hairpin RNA interference. In the XPC knocked-down cells, 100 {mu}M AZT treatment significantly increased [{sup 3}H]AZT incorporation into DNA, decreased the total number of viable cells, increased the release of lactate dehydrogenase, induced apoptosis, and caused a more extensive G2/M cell cycle arrest when compared to non-transfected HepG2 cells or HepG2 cells transfected with a scrambled short hairpin RNA sequence. Overall, these data indicate that XPC plays an essential role in the NER repair of AZT-induced DNA damage.
Heat shock protein 70 enhanced deoxyribonucleic acid base excision repair in human leukemic cells after ionizing radiation.
Bases, Robert
Base excision repair (BER) of DNA damage in irradiated THP1 human leukemic cells was stimulated by pretreating the cells with exogenous recombinant Hsp70. The treatment of THP1 cells with recombinant Hsp70 in cell culture promoted repair by reducing the frequency of apurinic, apyrimidinic (AP) sites in DNA before and after 1.3 Gy of radiation. However, by 30 minutes after 2.6 Gy, accelerated repair of abasic sites supervened, which may contribute to the loss of the very-low-dose cell hypersensitivity seen in clonogenic studies of other laboratories. After irradiation with 2.6 Gy, the crucial initial glycosylase step was markedly incomplete when cells had been transfected 24 hours before with a small interfering RNA (siRNA) designed to inhibit synthesis of Hsp70. In confirmation, lysates from irradiated siRNA-treated cells after 2.6 Gy were deficient in uracil glycosylase activity (UDG). Transfection with a scrambled RNA of the same size did not interfere with the glycosylase step, ie, the prompt conversion of damaged pyrimidine sites to abasic sites as well as the subsequent repair of those sites. BER measured by reduction of DNA AP sites before and after low-dose radiation was also deficient in THP1 cells that had been transfected with the siRNA designed to inhibit synthesis of Hsp70. These results implicate BER and the participation of Hsp70 in the repair of DNA in human leukemic cells with the doses of ionizing radiation used in clinical regimens. PMID:17009597
Effect of cordycepin(3'-deoxyadenosine) on excision repair of 5,6-dihydroxy-dihydrothymine-type products from the DNA of Micrococcus radiodurans
Patil, M.S.; Tundo, V.J.; Locher, S.E.; Hariharan, P.V.
Cordycepin(3'-deoxyadenosine), a nucleoside analog, has been shown to enhance radiation-induced cell killing. In an effort to elucidate the possible mechanism for enhancement of cell killing, the effect of cordycepin on the excision repair of radiation-induced 5,6-dihydroxy-dihydrothymine-type (t') products from the DNA of wild type Micrococcus radiodurans was investigated. The capacity of M. radiodurans to excise nondimeric (t') products from its DNA was significantly impaired after cordycepin treatment. The results suggest that the increased radiation sensitivity of cordycepin-treated cells could be due to alterations in cellular processes that repair DNA damage.
SIRT6 rescues the age related decline in base excision repair in a PARP1-dependent manner
Xu, Zhu; Zhang, Lei; Zhang, Wenjun; Meng, Du; Zhang, Hongxia; Jiang, Ying; Xu, Xiaojun; Van Meter, Michael; Seluanov, Andrei; Gorbunova, Vera; Mao, Zhiyong
In principle, a decline in base excision repair (BER) efficiency with age should lead to genomic instability and ultimately contribute to the onset of the aging phenotype. Although multiple studies have indicated a negative link between aging and BER, the change of BER efficiency with age in humans has not been systematically analyzed. Here, with foreskin fibroblasts isolated from 19 donors between 20 and 64 y of age, we report a significant decline of BER efficiency with age using a newly developed GFP reactivation assay. We further observed a very strong negative correlation between age and the expression levels of SIRT6, a factor which is known to maintain genomic integrity by improving DNA double strand break (DSB) repair. Our mechanistic study suggests that, similar to the regulatory role that SIRT6 plays in DNA DSB repair, SIRT6 regulates BER in a PARP1-depdendent manner. Moreover, overexpression of SIRT6 rescues the decline of BER in aged fibroblasts. In summary, our results uncovered the regulatory mechanisms of BER by SIRT6, suggesting that SIRT6 reactivation in aging tissues may help delay the process of aging through improving BER. PMID:25607651
A UV–Induced Genetic Network Links the RSC Complex to Nucleotide Excision Repair and Shows Dose-Dependent Rewiring
Srivas, Rohith; Costelloe, Thomas; Carvunis, Anne-Ruxandra; Sarkar, Sovan; Malta, Erik; Sun, Su Ming; Pool, Marijke; Licon, Katherine; van Welsem, Tibor; van Leeuwen, Fred; McHugh, Peter J.; van Attikum, Haico; Ideker, Trey
SUMMARY Efficient repair of UV-induced DNA damage requires the precise coordination of nucleotide excision repair (NER) with numerous other biological processes. To map this crosstalk, we generated a differential genetic interaction map centered on quantitative growth measurements of >45,000 double mutants before and after different doses of UV radiation. Integration of genetic data with physical interaction networks identified a global map of 89 UV-induced functional interactions amongst 62 protein complexes, including a number of links between the RSC complex and several NER factors. We show that RSC is recruited to both silenced and transcribed loci following UV damage where it facilitates efficient repair by promoting nucleosome remodeling. Finally, a comparison of the response to high versus low levels of UV shows that the degree of genetic rewiring correlates with dose of UV and reveals a network of dose-specific interactions. This study makes available a large resource of UV-induced interactions, and it illustrates a methodology for identifying dose-dependent interactions based on quantitative shifts in genetic networks. PMID:24360959
Enhanced Genotoxicity of Silver Nanoparticles in DNA Repair Deficient Mammalian Cells
Lim, Hui Kheng; Asharani, P. V.; Hande, M. Prakash
Silver nanoparticles (Ag-np) have been used in medicine and commercially due to their anti-microbial properties. Therapeutic potentials of these nanoparticles are being explored extensively despite the lack of information on their mechanism of action at molecular and cellular level. Here, we have investigated the DNA damage response and repair following Ag-np treatment in mammalian cells. Studies have shown that Ag-np exerts genotoxicity through double-strand breaks (DSBs). DNA-PKcs, the catalytic subunit of DNA dependent protein kinase, is an important caretaker of the genome which is known to be the main player mediating Non-homologous End-Joining (NHEJ) repair pathway. We hypothesize that DNA-PKcs is responsible for the repair of Ag-np induced DNA damage. In vitro studies have been carried out to investigate both cytotoxicity and genotoxicity induced by Ag-np in normal human cells, DNA-PKcs proficient, and deficient mammalian cells. Chemical inhibition of DNA-PKcs activity with NU7026, an ATP-competitive inhibitor of DNA-PKcs, has been performed to further validate the role of DNA-PKcs in this model. Our results suggest that Ag-np induced more prominent dose-dependent decrease in cell viability in DNA-PKcs deficient or inhibited cells. The deficiency or inhibition of DNA-PKcs renders the cells with higher susceptibility to DNA damage and genome instability which in turn contributed to greater cell cycle arrest/cell death. These findings support the fact that DNA-PKcs is involved in the repair of Ag-np induced genotoxicity and NHEJ repair pathway and DNA-PKcs particularly is activated to safeguard the genome upon Ag-np exposure. PMID:22707954
Structural Basis for Bulky-Adduct DNA-Lesion Recognition by the Nucleotide Excision Repair Protein Rad14.
Simon, Nina; Ebert, Charlotte; Schneider, Sabine
Heterocyclic aromatic amines react with purine bases and result in bulky DNA adducts that cause mutations. Such structurally diverse lesions are substrates for the nucleotide excision repair (NER). It is thought that the NER machinery recognises and verifies distorted DNA conformations, also involving the xeroderma pigmentosum group A and C proteins (XPA, XPC) that act as a scaffold between the DNA substrate and several other NER proteins. Here we present the synthesis of DNA molecules containing the polycyclic, aromatic amine C8-guanine lesions acetylaminophenyl, acetylaminonaphthyl, acetylaminoanthryl, and acetylaminopyrenyl, as well as their crystal structures in complex with the yeast XPA homologue Rad14. This work further substantiates the indirect lesion-detection mechanism employed by the NER system that recognises destabilised and deformable DNA structures. PMID:27223336
Structure and stereochemistry of the base excision repair glycosylase MutY reveal a mechanism similar to retaining glycosidases.
Woods, Ryan D; O'Shea, Valerie L; Chu, Aurea; Cao, Sheng; Richards, Jody L; Horvath, Martin P; David, Sheila S
MutY adenine glycosylases prevent DNA mutations by excising adenine from promutagenic 8-oxo-7,8-dihydroguanine (OG):A mismatches. Here, we describe structural features of the MutY active site bound to an azaribose transition state analog which indicate a catalytic role for Tyr126 and approach of the water nucleophile on the same side as the departing adenine base. The idea that Tyr126 participates in catalysis, recently predicted by modeling calculations, is strongly supported by mutagenesis and by seeing close contact between the hydroxyl group of this residue and the azaribose moiety of the transition state analog. NMR analysis of MutY methanolysis products corroborates a mechanism for adenine removal with retention of stereochemistry. Based on these results, we propose a revised mechanism for MutY that involves two nucleophilic displacement steps akin to the mechanisms accepted for 'retaining' O-glycosidases. This new-for-MutY yet familiar mechanism may also be operative in related base excision repair glycosylases and provides a critical framework for analysis of human MutY (MUTYH) variants associated with inherited colorectal cancer. PMID:26673696
Structure and stereochemistry of the base excision repair glycosylase MutY reveal a mechanism similar to retaining glycosidases
Woods, Ryan D.; O'Shea, Valerie L.; Chu, Aurea; Cao, Sheng; Richards, Jody L.; Horvath, Martin P.; David, Sheila S.
MutY adenine glycosylases prevent DNA mutations by excising adenine from promutagenic 8-oxo-7,8-dihydroguanine (OG):A mismatches. Here, we describe structural features of the MutY active site bound to an azaribose transition state analog which indicate a catalytic role for Tyr126 and approach of the water nucleophile on the same side as the departing adenine base. The idea that Tyr126 participates in catalysis, recently predicted by modeling calculations, is strongly supported by mutagenesis and by seeing close contact between the hydroxyl group of this residue and the azaribose moiety of the transition state analog. NMR analysis of MutY methanolysis products corroborates a mechanism for adenine removal with retention of stereochemistry. Based on these results, we propose a revised mechanism for MutY that involves two nucleophilic displacement steps akin to the mechanisms accepted for 'retaining' O-glycosidases. This new-for-MutY yet familiar mechanism may also be operative in related base excision repair glycosylases and provides a critical framework for analysis of human MutY (MUTYH) variants associated with inherited colorectal cancer. PMID:26673696
Silymarin Protects Epidermal Keratinocytes from Ultraviolet Radiation-Induced Apoptosis and DNA Damage by Nucleotide Excision Repair Mechanism
Katiyar, Santosh K.; Mantena, Sudheer K.; Meeran, Syed M.
Solar ultraviolet (UV) radiation is a well recognized epidemiologic risk factor for melanoma and non-melanoma skin cancers. This observation has been linked to the accumulation of UVB radiation-induced DNA lesions in cells, and that finally lead to the development of skin cancers. Earlier, we have shown that topical treatment of skin with silymarin, a plant flavanoid from milk thistle (Silybum marianum), inhibits photocarcinogenesis in mice; however it is less understood whether chemopreventive effect of silymarin is mediated through the repair of DNA lesions in skin cells and that protect the cells from apoptosis. Here, we show that treatment of normal human epidermal keratinocytes (NHEK) with silymarin blocks UVB-induced apoptosis of NHEK in vitro. Silymarin reduces the amount of UVB radiation-induced DNA damage as demonstrated by reduced amounts of cyclobutane pyrimidine dimers (CPDs) and as measured by comet assay, and that ultimately may lead to reduced apoptosis of NHEK. The reduction of UV radiation-induced DNA damage by silymarin appears to be related with induction of nucleotide excision repair (NER) genes, because UV radiation-induced apoptosis was not blocked by silymarin in NER-deficient human fibroblasts. Cytostaining and dot-blot analysis revealed that silymarin repaired UV-induced CPDs in NER-proficient fibroblasts from a healthy individual but did not repair UV-induced CPD-positive cells in NER-deficient fibroblasts from patients suffering from xeroderma pigmentosum complementation-A disease. Similarly, immunohistochemical analysis revealed that silymarin did not reduce the number of UVB-induced sunburn/apoptotic cells in the skin of NER-deficient mice, but reduced the number of sunburn cells in their wild-type counterparts. Together, these results suggest that silymarin exert the capacity to reduce UV radiation-induced DNA damage and, thus, prevent the harmful effects of UV radiation on the genomic stability of epidermal cells. PMID:21731736
Silymarin protects epidermal keratinocytes from ultraviolet radiation-induced apoptosis and DNA damage by nucleotide excision repair mechanism.
Katiyar, Santosh K; Mantena, Sudheer K; Meeran, Syed M
Solar ultraviolet (UV) radiation is a well recognized epidemiologic risk factor for melanoma and non-melanoma skin cancers. This observation has been linked to the accumulation of UVB radiation-induced DNA lesions in cells, and that finally lead to the development of skin cancers. Earlier, we have shown that topical treatment of skin with silymarin, a plant flavanoid from milk thistle (Silybum marianum), inhibits photocarcinogenesis in mice; however it is less understood whether chemopreventive effect of silymarin is mediated through the repair of DNA lesions in skin cells and that protect the cells from apoptosis. Here, we show that treatment of normal human epidermal keratinocytes (NHEK) with silymarin blocks UVB-induced apoptosis of NHEK in vitro. Silymarin reduces the amount of UVB radiation-induced DNA damage as demonstrated by reduced amounts of cyclobutane pyrimidine dimers (CPDs) and as measured by comet assay, and that ultimately may lead to reduced apoptosis of NHEK. The reduction of UV radiation-induced DNA damage by silymarin appears to be related with induction of nucleotide excision repair (NER) genes, because UV radiation-induced apoptosis was not blocked by silymarin in NER-deficient human fibroblasts. Cytostaining and dot-blot analysis revealed that silymarin repaired UV-induced CPDs in NER-proficient fibroblasts from a healthy individual but did not repair UV-induced CPD-positive cells in NER-deficient fibroblasts from patients suffering from xeroderma pigmentosum complementation-A disease. Similarly, immunohistochemical analysis revealed that silymarin did not reduce the number of UVB-induced sunburn/apoptotic cells in the skin of NER-deficient mice, but reduced the number of sunburn cells in their wild-type counterparts. Together, these results suggest that silymarin exert the capacity to reduce UV radiation-induced DNA damage and, thus, prevent the harmful effects of UV radiation on the genomic stability of epidermal cells. PMID:21731736
Both base excision repair and O6-methylguanine-DNA methyltransferase protect against methylation-induced colon carcinogenesis
Wirtz, Stefan; Nagel, Georg; Eshkind, Leonid; Neurath, Markus F.; Samson, Leona D.; Kaina, Bernd
Methylating agents are widely distributed environmental carcinogens. Moreover, they are being used in cancer chemotherapy. The primary target of methylating agents is DNA, and therefore, DNA repair is the first-line barrier in defense against their toxic and carcinogenic effects. Methylating agents induce in the DNA O6-methylguanine (O6MeG) and methylations of the ring nitrogens of purines. The lesions are repaired by O6-methylguanine-DNA methyltransferase (Mgmt) and by enzymes of the base excision repair (BER) pathway, respectively. Whereas O6MeG is well established as a pre-carcinogenic lesion, little is known about the carcinogenic potency of base N-alkylation products such as N3-methyladenine and N3-methylguanine. To determine their role in cancer formation and the role of BER in cancer protection, we checked the response of mice with a targeted gene disruption of Mgmt or N-alkylpurine-DNA glycosylase (Aag) or both Mgmt and Aag, to azoxymethane (AOM)-induced colon carcinogenesis, using non-invasive mini-colonoscopy. We demonstrate that both Mgmt- and Aag-null mice show a higher colon cancer frequency than the wild-type. With a single low dose of AOM (3 mg/kg) Aag-null mice showed an even stronger tumor response than Mgmt-null mice. The data provide evidence that both BER initiated by Aag and O6MeG reversal by Mgmt are required for protection against alkylation-induced colon carcinogenesis. Further, the data indicate that non-repaired N-methylpurines are not only pre-toxic but also pre-carcinogenic DNA lesions. PMID:20732909
Mechanisms of double-strand-break repair during gene targeting in mammalian cells.
Ng, P; Baker, M D
In the present study, the mechanism of double-strand-break (DSB) repair during gene targeting at the chromosomal immunoglobulin mu-locus in a murine hybridoma was examined. The gene-targeting assay utilized specially designed insertion vectors genetically marked in the region of homology to the chromosomal mu-locus by six diagnostic restriction enzyme site markers. The restriction enzyme markers permitted the contribution of vector-borne and chromosomal mu-sequences in the recombinant product to be determined. The use of the insertion vectors in conjunction with a plating procedure in which individual integrative homologous recombination events were retained for analysis revealed several important features about the mammalian DSB repair process:The presence of the markers within the region of shared homology did not affect the efficiency of gene targeting.In the majority of recombinants, the vector-borne marker proximal to the DSB was absent, being replaced with the corresponding chromosomal restriction enzyme site. This result is consistent with either formation and repair of a vector-borne gap or an "end" bias in mismatch repair of heteroduplex DNA (hDNA) that favored the chromosomal sequence. Formation of hDNA was frequently associated with gene targeting and, in most cases, began approximately 645 bp from the DSB and could encompass a distance of at least 1469 bp.The hDNA was efficiently repaired prior to DNA replication.The repair of adjacent mismatches in hDNA occurred predominantly on the same strand, suggesting the involvement of a long-patch repair mechanism. PMID:10049929
In vitro Repair of Oxidative DNA Damage by Human Nucleotide Excision Repair System: Possible Explanation for Neurodegeneration in Xeroderma Pigmentosum Patients
NASA Astrophysics Data System (ADS)
Reardon, Joyce T.; Bessho, Tadayoshi; Kung, Hsiang Chuan; Bolton, Philip H.; Sancar, Aziz
Xeroderma pigmentosum (XP) patients fail to remove pyrimidine dimers caused by sunlight and, as a consequence, develop multiple cancers in areas exposed to light. The second most common sign, present in 20-30% of XP patients, is a set of neurological abnormalities caused by neuronal death in the central and peripheral nervous systems. Neural tissue is shielded from sunlight-induced DNA damage, so the cause of neurodegeneration in XP patients remains unexplained. In this study, we show that two major oxidative DNA lesions, 8-oxoguanine and thymine glycol, are excised from DNA in vitro by the same enzyme system responsible for removing pyrimidine dimers and other bulky DNA adducts. Our results suggest that XP neurological disease may be caused by defective repair of lesions that are produced in nerve cells by reactive oxygen species generated as by-products of an active oxidative metabolism.
The role of Schizosaccharomyces pombe DNA repair enzymes Apn1p and Uve1p in the base excision repair of apurinic/apyrimidinic sites
Tanihigashi, Haruna; Yamada, Ayako; Igawa, Emi; Ikeda, Shogo . E-mail: email@example.com
In Schizosaccharomyces pombe the repair of apurinic/apyrimidinic (AP) sites is mainly initiated by AP lyase activity of DNA glycosylase Nth1p. In contrast, the major AP endonuclease Apn2p functions by removing 3'-{alpha},{beta}-unsaturated aldehyde ends induced by Nth1p, rather than by incising the AP sites. S. pombe possesses other minor AP endonuclease activities derived from Apn1p and Uve1p. In this study, we investigated the function of these two enzymes in base excision repair (BER) for methyl methanesulfonate (MMS) damage using the nth1 and apn2 mutants. Deletion of apn1 or uve1 from nth1{delta} cells did not affect sensitivity to MMS. Exogenous expression of Apn1p failed to suppress the MMS sensitivity of nth1{delta} cells. Although Apn1p and Uve1p incised the oligonucleotide containing an AP site analogue, these enzymes could not initiate repair of the AP sites in vivo. Despite this, expression of Apn1p partially restored the MMS sensitivity of apn2{delta} cells, indicating that the enzyme functions as a 3'-phosphodiesterase to remove 3'-blocked ends. Localization of Apn1p in the nucleus and cytoplasm hints at an additional function of the enzyme other than nuclear DNA repair. Heterologous expression of Saccharomyces cerevisiae homologue of Apn1p completely restored the MMS resistance of the nth1{delta} and apn2{delta} cells. This result confirms a difference in the major pathway for processing the AP site between S. pombe and S. cerevisiae cells.
The lung cancer mortality rate in Xuan Wei County is among the highest in China and has been attributed to exposure to indoor smoky coal emissions that contain very high levels of polycyclic aromatic hydrocarbons (PAHs). Nucleotide excision repair (NER) plays a key role in revers...
An Adenine-DNA Adduct Derived from Nitroreduction of 6-Nitrochrysene is more Resistant to Nucleotide Excision Repair than Guanine-DNA Adducts
Krzeminski, Jacek; Kropachev, Konstantin; Reeves, Dara; Kolbanovskiy, Aleksandr; Kolbanovskiy, Marina; Chen, Kun-Ming; Sharma, Arun K.; Geacintov, Nicholas; Amin, Shantu; El-Bayoumy, Karam
Previous studies in rats, mice and in vitro systems showed that 6-NC can be metabolically activated by two major pathways: 1) the formation of N-hydroxy-6-aminochrysene by nitroreduction to yield three major adducts: N-(dG-8-yl)-6-AC, 5-(dG-N2-yl)-6-AC and N-(dA-8-yl)-6-AC, and 2) the formation of trans-1,2-dihydroxy-1,2-dihydro-6-hydroxylaminochrysene (1,2-DHD-6-NHOH-C) by a combination of nitroreduction and ring oxidation pathways to yield: N-(dG-8-yl)-1,2-DHD-6-AC, 5-(dG-N2-yl)-1,2-DHD-6-AC and N-(dA-8-yl)-1,2-DHD-6-AC. These DNA lesions are likely to cause mutations if they are not removed by cellular defense mechanisms before DNA replication occurs. Here we compared for the first time, in HeLa cell extracts in vitro, the relative nucleotide excision repair (NER) efficiencies of DNA lesions derived from simple nitroreduction and from a combination of nitroreduction and ring oxidation pathways. We show that the N-(dG-8-yl)-1,2-DHD-6-AC adduct is more resistant to NER than the N-(dG-8-yl)-6-AC adduct by a factor of ~2. Furthermore, the N-(dA-8-yl)-6-AC is much more resistant to repair since its NER efficiency is ~ 8-fold lower than that of the N-(dG-8-yl)-6-AC adduct. On the basis of our previous study and the present investigation, lesions derived from 6-NC and benzo[a]pyrene can be ranked from the most to the least resistant lesion as follows: N-(dA-8-yl)-6-AC > N-(dG-8-yl)-1,2-DHD-6-AC > 5-(dG-N2-yl)-6-AC ~ N-(dG-8-yl)-6-AC ~ (+)-7R,8S,9S,10S-benzo[a]pyrene diol epoxide-derived trans-anti-benzo[a]pyrene-N2-dG adduct. The slow repair of the various lesions derived from 6-NC and thus their potential persistence in mammalian tissue, could in part account for the powerful carcinogenicity of 6-NC as compared to B[a]P in the rat mammary gland. PMID:24112095
Excision repair of UV-induced pyrimidine dimers in human skin in vivo
D'Ambrosio, S.M.; Slazinski, L.; Whetstone, J.W.; Lowney, E.
The induction and loss of pyrimidine dimers in human skin in vivo was determined using UV endonuclease, alkaline sucrose sedimentations, and the fluorescent detection of nonradiolabeled DNA. The number of dimers induced following exposure of the skin to radiation emitted from a Burdick UV-800 sunlamp was quantitated by reacting the extracted DNA with Micrococcus luteus endonuclease specific for pyrimidine dimers. Exposure to 15 and 30 seconds of radiation emitted from this lamp produced the formation of 12.8 and 23.6 dimers per 10(8) daltons DNA, respectively. Approximately 50% of the dimers induced were lost 58 min after irradiation. Only a small percentage (less than 10) remained 24 hr postirradiation. These data partially characterize the process by which pyrimidine dimers are excised from human skin DNA in vivo.
Poly(ADP-ribose)-synthesis and excision repair in light sensitive skin disorders.
Horkay, I; Topaloglou, A; Teherani, D K; Kósa, A; Altmann, H
Several data suggest a relationship of poly(ADP-ribose) (PAR) synthesis to DNA repair and the influence of some trace elements on the semiconservative and unscheduled DNA synthesis (UDS). Previously we found certain alterations in the UV-light induced UDS and in the contents of trace elements in the lymphocytes of patients with light sensitive skin disorders. In the recent study in polymorphic light eruption, cutaneous porphyrias and xeroderma pigmentosum the PAR synthesis and zinc, copper and manganese contents in the chromatin of the lymphocytes (measured by neutron activation analysis) were investigated. UV induced PAR synthesis was generally lower in the cells of polymorphic light eruption and especially in xeroderma pigmentosum with a reduced repair capacity whereas in cutaneous porphyrias no difference was observed. Some correlations occurred between the contents of trace elements studied and UDS as well in each group tested. It seems that PAR investigations throw new light upon our understanding of the pathomechanism of photodermatoses. PMID:2094134
Calcium-Binding Capacity of Centrin2 Is Required for Linear POC5 Assembly but Not for Nucleotide Excision Repair
Dantas, Tiago J.; Daly, Owen M.; Conroy, Pauline C.; Tomas, Martin; Wang, Yifan; Lalor, Pierce; Dockery, Peter; Ferrando-May, Elisa; Morrison, Ciaran G.
Centrosomes, the principal microtubule-organising centres in animal cells, contain centrins, small, conserved calcium-binding proteins unique to eukaryotes. Centrin2 binds to xeroderma pigmentosum group C protein (XPC), stabilising it, and its presence slightly increases nucleotide excision repair (NER) activity in vitro. In previous work, we deleted all three centrin isoforms present in chicken DT40 cells and observed delayed repair of UV-induced DNA lesions, but no centrosome abnormalities. Here, we explore how centrin2 controls NER. In the centrin null cells, we expressed centrin2 mutants that cannot bind calcium or that lack sites for phosphorylation by regulatory kinases. Expression of any of these mutants restored the UV sensitivity of centrin null cells to normal as effectively as expression of wild-type centrin. However, calcium-binding-deficient and T118A mutants showed greatly compromised localisation to centrosomes. XPC recruitment to laser-induced UV-like lesions was only slightly slower in centrin-deficient cells than in controls, and levels of XPC and its partner HRAD23B were unaffected by centrin deficiency. Interestingly, we found that overexpression of the centrin interactor POC5 leads to the assembly of linear, centrin-dependent structures that recruit other centrosomal proteins such as PCM-1 and NEDD1. Together, these observations suggest that assembly of centrins into complex structures requires calcium binding capacity, but that such assembly is not required for centrin activity in NER. PMID:23844208
Neil3-dependent base excision repair regulates lipid metabolism and prevents atherosclerosis in Apoe-deficient mice
Skarpengland, Tonje; Holm, Sverre; Scheffler, Katja; Gregersen, Ida; Dahl, Tuva B.; Suganthan, Rajikala; Segers, Filip M.; Østlie, Ingunn; Otten, Jeroen J. T.; Luna, Luisa; Ketelhuth, Daniel F. J.; Lundberg, Anna M.; Neurauter, Christine G.; Hildrestrand, Gunn; Skjelland, Mona; Bjørndal, Bodil; Svardal, Asbjørn M.; Iversen, Per O.; Hedin, Ulf; Nygård, Ståle; Olstad, Ole K.; Krohg-Sørensen, Kirsten; Slupphaug, Geir; Eide, Lars; Kuśnierczyk, Anna; Folkersen, Lasse; Ueland, Thor; Berge, Rolf K.; Hansson, Göran K.; Biessen, Erik A. L.; Halvorsen, Bente; Bjørås, Magnar; Aukrust, Pål
Increasing evidence suggests that oxidative DNA damage accumulates in atherosclerosis. Recently, we showed that a genetic variant in the human DNA repair enzyme NEIL3 was associated with increased risk of myocardial infarction. Here, we explored the role of Neil3/NEIL3 in atherogenesis by both clinical and experimental approaches. Human carotid plaques revealed increased NEIL3 mRNA expression which significantly correlated with mRNA levels of the macrophage marker CD68. Apoe−/−Neil3−/− mice on high-fat diet showed accelerated plaque formation as compared to Apoe−/− mice, reflecting an atherogenic lipid profile, increased hepatic triglyceride levels and attenuated macrophage cholesterol efflux capacity. Apoe−/−Neil3−/− mice showed marked alterations in several pathways affecting hepatic lipid metabolism, but no genotypic alterations in genome integrity or genome-wide accumulation of oxidative DNA damage. These results suggest a novel role for the DNA glycosylase Neil3 in atherogenesis in balancing lipid metabolism and macrophage function, potentially independently of genome-wide canonical base excision repair of oxidative DNA damage. PMID:27328939
Deficiency in nucleotide excision repair family gene activity, especially ERCC3, is associated with non-pigmented hair fiber growth.
Yu, Mei; Bell, Robert H; Ho, Maggie M; Leung, Gigi; Haegert, Anne; Carr, Nicholas; Shapiro, Jerry; McElwee, Kevin J
We conducted a microarray study to discover gene expression patterns associated with a lack of melanogenesis in non-pigmented hair follicles (HF) by microarray. Pigmented and non-pigmented HFs were collected and micro-dissected into the hair bulb (HB) and the upper hair sheaths (HS) including the bulge region. In comparison to pigmented HS and HBs, nucleotide excision repair (NER) family genes ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, ERCC6, XPA, NTPBP, HCNP, DDB2 and POLH exhibited statistically significantly lower expression in non- pigmented HS and HBs. Quantitative PCR verified microarray data and identified ERCC3 as highly differentially expressed. Immunohistochemistry confirmed ERCC3 expression in HF melanocytes. A reduction in ERCC3 by siRNA interference in human melanocytes in vitro reduced their tyrosinase production ability. Our results suggest that loss of NER gene function is associated with a loss of melanin production capacity. This may be due to reduced gene transcription and/or reduced DNA repair in melanocytes which may eventually lead to cell death. These results provide novel information with regard to melanogenesis and its regulation. PMID:22615732
The Potential Role of 8-Oxoguanine DNA Glycosylase-Driven DNA Base Excision Repair in Exercise-Induced Asthma
Belanger, KarryAnne K.; Ameredes, Bill T.; Boldogh, Istvan
Asthma is characterized by reversible airway narrowing, shortness of breath, wheezing, coughing, and other symptoms driven by chronic inflammatory processes, commonly triggered by allergens. In 90% of asthmatics, most of these symptoms can also be triggered by intense physical activities and severely exacerbated by environmental factors. This condition is known as exercise-induced asthma (EIA). Current theories explaining EIA pathogenesis involve osmotic and/or thermal alterations in the airways caused by changes in respiratory airflow during exercise. These changes, along with existing airway inflammatory conditions, are associated with increased cellular levels of reactive oxygen species (ROS) affecting important biomolecules including DNA, although the underlying molecular mechanisms have not been completely elucidated. One of the most abundant oxidative DNA lesions is 8-oxoguanine (8-oxoG), which is repaired by 8-oxoguanine DNA glycosylase 1 (OGG1) during the base excision repair (BER) pathway. Whole-genome expression analyses suggest a cellular response to OGG1-BER, involving genes that may have a role in the pathophysiology of EIA leading to mast cell degranulation, airway hyperresponsiveness, and bronchoconstriction. Accordingly, this review discusses a potential new hypothesis in which OGG1-BER-induced gene expression is associated with EIA symptoms. PMID:27524866
Quantitative, real-time analysis of base excision repair activity in cell lysates utilizing lesion-specific molecular beacons.
Svilar, David; Vens, Conchita; Sobol, Robert W
We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via Förster Resonance Energy Transfer (FRET). The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors. PMID:22895410
Base excision repair of ionizing radiation-induced DNA damage in G1 and G2 cell cycle phases
Chaudhry, M Ahmad
Background Major genomic surveillance mechanisms regulated in response to DNA damage exist at the G1/S and G2/M checkpoints. It is presumed that these delays provide time for the repair of damaged DNA. Cells have developed multiple DNA repair pathways to protect themselves from different types of DNA damage. Oxidative DNA damage is processed by the base excision repair (BER) pathway. Little is known about the BER of ionizing radiation-induced DNA damage and putative heterogeneity of BER in the cell cycle context. We measured the activities of three BER enzymes throughout the cell cycle to investigate the cell cycle-specific repair of ionizing radiation-induced DNA damage. We further examined BER activities in G2 arrested human cells after exposure to ionizing radiation. Results Using an in vitro incision assay involving radiolabeled oligonucleotides with specific DNA lesions, we examined the activities of several BER enzymes in the whole cell extracts prepared from synchronized human HeLa cells irradiated in G1 and G2 phase of the cell cycle. The activities of human endonuclease III (hNTH1), a glycosylase/lyase that removes several damaged bases from DNA including dihydrouracil (DHU), 8-oxoguanine-DNA glycosylase (hOGG1) that recognizes 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG) lesion and apurinic/apyrimidinic endonuclease (hAPE1) that acts on abasic sites including synthetic analog furan were examined. Conclusion Overall the repair activities of hNTH1 and hAPE1 were higher in the G1 compared to G2 phase of the cell cycle. The percent cleavages of oligonucleotide substrate with furan were greater than substrate with DHU in both G1 and G2 phases. The irradiation of cells enhanced the cleavage of substrates with furan and DHU only in G1 phase. The activity of hOGG1 was much lower and did not vary within the cell cycle. These results demonstrate the cell cycle phase dependence on the BER of ionizing radiation-induced DNA damage. Interestingly no evidence of
Identification of a conserved 5'-dRP lyase activity in bacterial DNA repair ligase D and its potential role in base excision repair.
de Ory, Ana; Nagler, Katja; Carrasco, Begoña; Raguse, Marina; Zafra, Olga; Moeller, Ralf; de Vega, Miguel
Bacillus subtilis is one of the bacterial members provided with a nonhomologous end joining (NHEJ) system constituted by the DNA-binding Ku homodimer that recruits the ATP-dependent DNA Ligase D (BsuLigD) to the double-stranded DNA breaks (DSBs) ends. BsuLigD has inherent polymerization and ligase activities that allow it to fill the short gaps that can arise after realignment of the broken ends and to seal the resulting nicks, contributing to genome stability during the stationary phase and germination of spores. Here we show that BsuLigD also has an intrinsic 5'-2-deoxyribose-5-phosphate (dRP) lyase activity located at the N-terminal ligase domain that in coordination with the polymerization and ligase activities allows efficient repairing of 2'-deoxyuridine-containing DNA in an in vitro reconstituted Base Excision Repair (BER) reaction. The requirement of a polymerization, a dRP removal and a final sealing step in BER, together with the joint participation of BsuLigD with the spore specific AP endonuclease in conferring spore resistance to ultrahigh vacuum desiccation suggest that BsuLigD could actively participate in this pathway. We demonstrate the presence of the dRP lyase activity also in the homolog protein from the distantly related bacterium Pseudomonas aeruginosa, allowing us to expand our results to other bacterial LigDs. PMID:26826709
Identification of a conserved 5′-dRP lyase activity in bacterial DNA repair ligase D and its potential role in base excision repair
de Ory, Ana; Nagler, Katja; Carrasco, Begoña; Raguse, Marina; Zafra, Olga; Moeller, Ralf; de Vega, Miguel
Bacillus subtilis is one of the bacterial members provided with a nonhomologous end joining (NHEJ) system constituted by the DNA-binding Ku homodimer that recruits the ATP-dependent DNA Ligase D (BsuLigD) to the double-stranded DNA breaks (DSBs) ends. BsuLigD has inherent polymerization and ligase activities that allow it to fill the short gaps that can arise after realignment of the broken ends and to seal the resulting nicks, contributing to genome stability during the stationary phase and germination of spores. Here we show that BsuLigD also has an intrinsic 5′-2-deoxyribose-5-phosphate (dRP) lyase activity located at the N-terminal ligase domain that in coordination with the polymerization and ligase activities allows efficient repairing of 2′-deoxyuridine-containing DNA in an in vitro reconstituted Base Excision Repair (BER) reaction. The requirement of a polymerization, a dRP removal and a final sealing step in BER, together with the joint participation of BsuLigD with the spore specific AP endonuclease in conferring spore resistance to ultrahigh vacuum desiccation suggest that BsuLigD could actively participate in this pathway. We demonstrate the presence of the dRP lyase activity also in the homolog protein from the distantly related bacterium Pseudomonas aeruginosa, allowing us to expand our results to other bacterial LigDs. PMID:26826709
DNA polymerase X from Deinococcus radiodurans implicated in bacterial tolerance to DNA damage is characterized as a short patch base excision repair polymerase.
Khairnar, Nivedita P; Misra, Hari S
The Deinococcus radiodurans R1 genome encodes an X-family DNA repair polymerase homologous to eukaryotic DNA polymerase beta. The recombinant deinococcal polymerase X (PolX) purified from transgenic Escherichia coli showed deoxynucleotidyltransferase activity. Unlike the Klenow fragment of E. coli, this enzyme showed short patch DNA synthesis activity on heteropolymeric DNA substrate. The recombinant enzyme showed 5'-deoxyribose phosphate (5'-dRP) lyase activity and base excision repair function in vitro, with the help of externally supplied glycosylase and AP endonuclease functions. A polX disruption mutant of D. radiodurans expressing 5'-dRP lyase and a truncated polymerase domain was comparatively less sensitive to gamma-radiation than a polX deletion mutant. Both mutants showed higher sensitivity to hydrogen peroxide. Excision repair mutants of E. coli expressing this polymerase showed functional complementation of UV sensitivity. These results suggest the involvement of deinococcal polymerase X in DNA-damage tolerance of D. radiodurans, possibly by contributing to DNA double-strand break repair and base excision repair. PMID:19542005
8-oxoguanine DNA glycosylase-1 driven DNA base excision repair: role in asthma pathogenesis
Ba, Xueqing; Aguilera Aguirre, Leopoldo; Sur, Sanjiv; Boldogh, Istvan
Purpose of review To provide both an overview and evidence of the potential etiology of oxidative DNA base damage and repair-signaling in chronic inflammation and histological changes associated with asthma. Recent findings Asthma is initiated/maintained by immunological, genetic/epigenetic and environmental factors. It is a world-wide health problem, as current therapies suppress symptoms rather than prevent/reverse the disease, largely due to gaps in understanding its molecular mechanisms. Inflammation, oxidative stress and DNA damage are inseparable phenomena, but their molecular roles in asthma pathogenesis are unclear. It was found that among oxidatively modified DNA bases, 8-oxoguanine (8-oxoG) is one of the most abundant, and its levels in DNA and body fluids are considered a biomarker of ongoing asthmatic processes. Free 8-oxoG forms a complex with 8-oxoguanine DNA glycosylase-1 (OGG1) and activates RAS-family GTPases that induce gene expression to mobilize innate and adaptive immune systems, along with genes regulating airway hyperplasia, hyper-responsiveness and lung remodeling in atopic and non-atopic asthma. Summary DNA's integrity must be maintained to prevent mutation, so its continuous repair and downstream signaling "fuels" chronic inflammatory processes in asthma, and forms the basic mechanism whose elucidation will allow the development of new drug targets for the prevention/reversal of lung diseases. PMID:25486379
UvrD Participation in Nucleotide Excision Repair Is Required for the Recovery of DNA Synthesis following UV-Induced Damage in Escherichia coli.
Newton, Kelley N; Courcelle, Charmain T; Courcelle, Justin
UvrD is a DNA helicase that participates in nucleotide excision repair and several replication-associated processes, including methyl-directed mismatch repair and recombination. UvrD is capable of displacing oligonucleotides from synthetic forked DNA structures in vitro and is essential for viability in the absence of Rep, a helicase associated with processing replication forks. These observations have led others to propose that UvrD may promote fork regression and facilitate resetting of the replication fork following arrest. However, the molecular activity of UvrD at replication forks in vivo has not been directly examined. In this study, we characterized the role UvrD has in processing and restoring replication forks following arrest by UV-induced DNA damage. We show that UvrD is required for DNA synthesis to recover. However, in the absence of UvrD, the displacement and partial degradation of the nascent DNA at the arrested fork occur normally. In addition, damage-induced replication intermediates persist and accumulate in uvrD mutants in a manner that is similar to that observed in other nucleotide excision repair mutants. These data indicate that, following arrest by DNA damage, UvrD is not required to catalyze fork regression in vivo and suggest that the failure of uvrD mutants to restore DNA synthesis following UV-induced arrest relates to its role in nucleotide excision repair. PMID:23056919
Caloric restriction promotes genomic stability by induction of base excision repair and reversal of its age-related decline.
Cabelof, Diane C; Yanamadala, Sunitha; Raffoul, Julian J; Guo, ZhongMao; Soofi, Abdulsalam; Heydari, Ahmad R
Caloric restriction is a potent experimental manipulation that extends mean and maximum life span and delays the onset and progression of tumors in laboratory rodents. While caloric restriction (CR) clearly protects the genome from deleterious damage, the mechanism by which genomic stability is achieved remains unclear. We provide evidence that CR promotes genomic stability by increasing DNA repair capacity, specifically base excision repair (BER). CR completely reverses the age-related decline in BER capacity (P<0.01) in all tissues tested (brain, liver, spleen and testes) providing aged, CR animals with the BER phenotype of young, ad libitum-fed animals. This CR-induced reversal of the aged BER phenotype is accompanied by a reversal in the age-related decline in DNA polymerase beta (beta-pol), a rate-limiting enzyme in the BER pathway. CR significantly reversed the age-related loss of beta-pol protein levels (P<0.01), mRNA levels (P<0.01) and enzyme activity (P<0.01) in all tissues tested. Additionally, in young (4-6-month-old) CR animals a significant up-regulation in BER capacity, beta-pol protein and beta-pol mRNA is observed (P<0.01), demonstrating an early effect of CR that may provide insight in distinguishing the anti-tumor from the anti-aging effects of CR. This up-regulation in BER by caloric restriction in young animals corresponds to increased protection from carcinogen exposure, as mutation frequency is significantly reduced in CR animals exposed to either DMS or 2-nitropropane (2-NP) (P<0.01). Overall the data suggest an important biological consequence of moderate BER up-regulation and provides support for the hormesis theory of caloric restriction. PMID:12547392
Inter-individual variation in nucleotide excision repair in young adults: effects of age, adiposity, micronutrient supplementation and genotype.
Tyson, John; Caple, Fiona; Spiers, Alison; Burtle, Brian; Daly, Ann K; Williams, Elizabeth A; Hesketh, John E; Mathers, John C
Nucleotide excision repair (NER) is responsible for repairing bulky helix-distorting DNA lesions and is essential for the maintenance of genomic integrity. Severe hereditary impairment of NER leads to cancers such as those in xeroderma pigmentosum, and more moderate reductions in NER capacity have been associated with an increased cancer risk. Diet is a proven modifier of cancer risk but few studies have investigated the potential relationships between diet and NER. In the present study, the plasmid-based host cell reactivation assay was used to measure the NER capacity in peripheral blood mononuclear cells from fifty-seven volunteers aged 18-30 years before and after 6 weeks of supplementation with micronutrients (selenium and vitamins A, C and E). As a control, nine individuals remained unsupplemented over the same period. Volunteers were genotyped for the following polymorphisms in NER genes: ERCC5 Asp1104His (rs17655); XPC Lys939Gln (rs2228001); ERCC2 Lys751Gnl (rs13181); XPC PAT (an 83 bp poly A/T insertion-deletion polymorphism in the XPC gene). NER capacity varied 11-fold between individuals and was inversely associated with age and endogenous DNA strand breaks. For the first time, we observed an inverse association between adiposity and NER. No single polymorphism was associated with the NER capacity, although significant gene-gene interactions were observed between XPC Lys939Gln and ERCC5 Asp1104His and XPC Lys939Gln and ERCC2 Lys751Gnl. While there was no detectable effect of micronutrient supplementation on NER capacity, there was evidence that the effect of fruit intake on the NER capacity may be modulated by the ERCC2 Lys751Gnl single nucleotide polymorphism. PMID:18838045
Twist-open mechanism of DNA damage recognition by the Rad4/XPC nucleotide excision repair complex
Velmurugu, Yogambigai; Chen, Xuejing; Slogoff Sevilla, Phillip; Min, Jung-Hyun; Ansari, Anjum
DNA damage repair starts with the recognition of damaged sites from predominantly normal DNA. In eukaryotes, diverse DNA lesions from environmental sources are recognized by the xeroderma pigmentosum C (XPC) nucleotide excision repair complex. Studies of Rad4 (radiation-sensitive 4; yeast XPC ortholog) showed that Rad4 "opens" up damaged DNA by inserting a β-hairpin into the duplex and flipping out two damage-containing nucleotide pairs. However, this DNA lesion "opening" is slow (˜5–10 ms) compared with typical submillisecond residence times per base pair site reported for various DNA-binding proteins during 1D diffusion on DNA. To address the mystery as to how Rad4 pauses to recognize lesions during diffusional search, we examine conformational dynamics along the lesion recognition trajectory using temperature-jump spectroscopy. Besides identifying the ˜10-ms step as the rate-limiting bottleneck towards opening specific DNA site, we uncover an earlier ˜100- to 500-μs step that we assign to nonspecific deformation (unwinding/"twisting") of DNA by Rad4. The β-hairpin is not required to unwind or to overcome the bottleneck but is essential for full nucleotide-flipping. We propose that Rad4 recognizes lesions in a step-wise "twist-open" mechanism, in which preliminary twisting represents Rad4 interconverting between search and interrogation modes. Through such conformational switches compatible with rapid diffusion on DNA, Rad4 may stall preferentially at a lesion site, offering time to open DNA. This study represents the first direct observation, to our knowledge, of dynamical DNA distortions during search/interrogation beyond base pair breathing. Submillisecond interrogation with preferential stalling at cognate sites may be common to various DNA-binding proteins. PMID:27035942
Nucleotide Excision Repair Factor XPC Enhances DNA Damage-Induced Apoptosis by Downregulating the Antiapoptotic Short Isoform of Caspase-2
Wang, Qi-En; Han, Chunhua; Zhang, Bo; Sabapathy, Kanaga; Wani, Altaf A.
XPC protein is a critical DNA damage recognition factor in nucleotide excision repair (NER) for which genetic deficiency confers a predisposition to cancer. In this study we demonstrate that XPC has a function that is independent of its canonical function in DNA repair, potentially altering the interpretation of how XPC deficiency leads to heightened cancer susceptibility. XPC enhances apoptosis induced by DNA damage in a p53 nullizygous background, acting downstream of mitochondrial permeabilization and upstream of caspase-9 activation in the DNA damage-induced apoptosis cascade. We found that deficiency in XPC upregulated production of the short isoform of caspase-2 (casp-2S). This upregulation occurred at both protein and mRNA levels through repression of the caspase-2 promoter by XPC protein. Targeted RNAi-mediated downregulation of casp-2S enhanced UV-induced apoptosis as well as activation of caspase-9 and caspase-6 in XPC-deficient cells, but not in XPC-proficient cells. In addition, XPC overexpression in various p53-deficient cancer cells resistant to cisplatin improved their sensitivity to cisplatin-induced apoptosis. Given that casp-2S functions as an anti-apoptotic protein, our findings suggest that XPC enhances DNA damage-induced apoptosis through inhibition of casp-2S transcription. Together, these findings offer a mechanistic foundation to overcome the resistance of highly prevalent p53-deficient tumors to cell death induced by DNA-damaging therapeutic agents, by targeting strategies that inhibit the expression or function of casp-2S. PMID:22174370
Twist-open mechanism of DNA damage recognition by the Rad4/XPC nucleotide excision repair complex.
Velmurugu, Yogambigai; Chen, Xuejing; Slogoff Sevilla, Phillip; Min, Jung-Hyun; Ansari, Anjum
DNA damage repair starts with the recognition of damaged sites from predominantly normal DNA. In eukaryotes, diverse DNA lesions from environmental sources are recognized by the xeroderma pigmentosum C (XPC) nucleotide excision repair complex. Studies of Rad4 (radiation-sensitive 4; yeast XPC ortholog) showed that Rad4 "opens" up damaged DNA by inserting a β-hairpin into the duplex and flipping out two damage-containing nucleotide pairs. However, this DNA lesion "opening" is slow (˜5-10 ms) compared with typical submillisecond residence times per base pair site reported for various DNA-binding proteins during 1D diffusion on DNA. To address the mystery as to how Rad4 pauses to recognize lesions during diffusional search, we examine conformational dynamics along the lesion recognition trajectory using temperature-jump spectroscopy. Besides identifying the ˜10-ms step as the rate-limiting bottleneck towards opening specific DNA site, we uncover an earlier ˜100- to 500-μs step that we assign to nonspecific deformation (unwinding/"twisting") of DNA by Rad4. The β-hairpin is not required to unwind or to overcome the bottleneck but is essential for full nucleotide-flipping. We propose that Rad4 recognizes lesions in a step-wise "twist-open" mechanism, in which preliminary twisting represents Rad4 interconverting between search and interrogation modes. Through such conformational switches compatible with rapid diffusion on DNA, Rad4 may stall preferentially at a lesion site, offering time to open DNA. This study represents the first direct observation, to our knowledge, of dynamical DNA distortions during search/interrogation beyond base pair breathing. Submillisecond interrogation with preferential stalling at cognate sites may be common to various DNA-binding proteins. PMID:27035942
Post-UV survival and mutagenesis in DNA repair-proficient and -deficient strains of Escherichia coli K-12 grown in 5-azacytidine to inhibit DNA cytosine methylation: evidence for mutagenic excision repair.
Radnedge, L; Pinney, R J
Inhibition of cytosine methylation by growth in 5-azacytidine (5-azaC), did not affect the sensitivities to DNA damage induced by exposure to ultraviolet light (UV) of Escherichia coli K-12 strains AB1157 dcm+, which is fully DNA repair-proficient, LR68 (a dcm derivative of AB1157), JC3890 dcm+ uvrB, deficient in error-free excision repair, TK702 dcm+ umuC, deficient in error-prone repair, or TK501 dcm+ uvrB umuC, which lacks both excision repair and error-prone repair. However, growth in 5-azaC increased the post-UV survival of strains AB2463 recA(Def), AB2470 recB and AB2494 lexA(Ind-), which are deficient in the induction or expression of recombination repair or error-prone repair of DNA. Spontaneous mutation frequencies were increased in strains LR68, AB2463, AB2470 and AB2494 by growth in 5-azaC, but remained unaltered in strains AB1157, JC3890, TK702 or TK501. Growth in 5-azaC significantly increased UV-induced mutation frequencies in strains AB2463 and AB2470, significantly reduced UV-induced mutation in strain JC3890, but had little effect on UV-induced mutation in the other strains. The results suggest that 5-azaC may induce a normally error-free DNA repair pathway to become error-prone and therefore genotoxic. PMID:7683337
Up-regulation of nucleotide excision repair in mouse lung and liver following chronic exposure to aflatoxin B{sub 1} and its dependence on p53 genotype
Mulder, Jeanne E.; Bondy, Genevieve S.; Mehta, Rekha; Massey, Thomas E.
Aflatoxin B{sub 1} (AFB{sub 1}) is biotransformed in vivo into an epoxide metabolite that forms DNA adducts that may induce cancer if not repaired. p53 is a tumor suppressor gene implicated in the regulation of global nucleotide excision repair (NER). Male heterozygous p53 knockout (B6.129-Trp53{sup tm1Brd}N5, Taconic) and wild-type mice were exposed to 0, 0.2 or 1.0 ppm AFB{sub 1} for 26 weeks. NER activity was assessed with an in vitro assay, using AFB{sub 1}-epoxide adducted plasmid DNA as a substrate. For wild-type mice, repair of AFB{sub 1}–N7-Gua adducts was 124% and 96% greater in lung extracts from mice exposed to 0.2 ppm and 1.0 ppm AFB{sub 1} respectively, and 224% greater in liver extracts from mice exposed to 0.2 ppm AFB{sub 1} (p < 0.05). In heterozygous p53 knockout mice, repair of AFB{sub 1}–N7-Gua was only 45% greater in lung extracts from mice exposed to 0.2 ppm AFB{sub 1} (p < 0.05), and no effect was observed in lung extracts from mice treated with 1.0 ppm AFB{sub 1} or in liver extracts from mice treated with either AFB{sub 1} concentration. p53 genotype did not affect basal levels of repair. AFB{sub 1} exposure did not alter repair of AFB{sub 1}-derived formamidopyrimidine adducts in lung or liver extracts of either mouse genotype nor did it affect XPA or XPB protein levels. In summary, chronic exposure to AFB{sub 1} increased NER activity in wild-type mice, and this response was diminished in heterozygous p53 knockout mice, indicating that loss of one allele of p53 limits the ability of NER to be up-regulated in response to DNA damage. - Highlights: • Mice are chronically exposed to low doses of the mycotoxin aflatoxin B{sub 1} (AFB{sub 1}). • The effects of AFB{sub 1} and p53 status on nucleotide excision repair are investigated. • AFB{sub 1} increases nucleotide excision repair in wild type mouse lung and liver. • This increase is attenuated in p53 heterozygous mouse lung and liver. • Results portray the role of p53 in
Expression of Excision Repair Cross-Complementation Group 1 as Predictive Marker for Nasopharyngeal Cancer Treated With Concurrent Chemoradiotherapy
Sun, Jong-Mu; Ahn, Myung-Ju; Park, Min Jae; Lee, Hui-Young; Ahn, Jin Seok; Lee, Seungkoo; Kang, Gu; Han, Joungho; Son, Young-Ik; Baek, Chung-Hwan; Ahn, Yong Chan; Park, Keunchil
Purpose: Cisplatin-based concurrent chemoradiotherapy is the standard treatment of nasopharyngeal cancer. The expression of excision repair cross-complementation group 1 (ERCC1) has been reported to be associated with resistance to platinum-based chemotherapy. We evaluated whether ERCC1 expression could predict the treatment response and survival outcome of patients with locally advanced nasopharyngeal cancer who were treated with cisplatin-based concurrent chemoradiotherapy. Methods and Materials: Immunohistochemistry was used to examine the expression of ERCC1 in nasopharyngeal tumor tissue. Patients were categorized into either a resistant or sensitive group depending on their treatment response outcome. A total of 77 patients were assessed in the present study. Results: The resistant and sensitive groups included 25 and 52 patients, respectively. ERCC1 expression was positive in the tumor tissue for 39 of the 77 patients (51%). Significantly more ERCC1-negative tumors were in the sensitive group than in the resistant group (p = .035). In terms of survival outcome, univariate analysis determined that patients with ERCC1-negative tumors had longer disease-free survival (p = .076) and overall survival (p = .013) than patients with ERCC1-positive tumors. Multivariate analysis determined that negative ERCC expression in tumors was an independent predictor for prolonged overall survival (hazard ratio, 0.14; 95% confidence interval, 0.03-0.71). Conclusion: These results suggest that ERCC1 expression might be a useful predictive marker in patients with locally advanced nasopharyngeal cancer who are under consideration for cisplatin-based concurrent chemoradiotherapy.
Heterogeneity of excision repair cross-complementation group 1 gene expression in non-small-cell lung cancer patients
Excision repair cross-complementation group 1 (ERCC1) gene expression analysis is currently used widely in the molecular diagnosis of cancer. According to numerous studies, ERCC1 gene expression correlates with overall survival and effectiveness of chemotherapy with platinum agents. However, the degree of this correlation differs among various studies, with certain authors reporting a complete lack of such a correlation. These contradictions may be attributed to a number of factors, including the heterogeneity of the tumor tissue. In this study, we attempted to assess the degree of genetic heterogeneity exhibited by tissue samples obtained from non-small-cell lung cancer (NSCLC) through the expression of the ERCC1 gene. This study included 25 samples of tumor tissue from patients with a morphologically confirmed NSCLC diagnosis. A total of three randomized sections of each specimen were used. The ERCC1 gene expression was assessed by quantitative polymerase chain reaction (qPCR) in the TaqMan format. When planning the experiment and analysis of qPCR data, the MIQE guidelines were taken into consideration. We established that the coefficient of variation of the relative level of ERCC1 gene expression in the majority of the samples exceeded 33% (P<0.05), indicating the significant heterogeneity of the sample. We also demonstrated that the degree of heterogeneity of the tumor tissue is largely dependent on disease stage. PMID:25469300
Structure-function analysis of the EF-hand protein centrin-2 for its intracellular localization and nucleotide excision repair
Nishi, Ryotaro; Sakai, Wataru; Tone, Daisuke; Hanaoka, Fumio; Sugasawa, Kaoru
Centrin-2 is an evolutionarily conserved, calmodulin-related protein, which is involved in multiple cellular functions including centrosome regulation and nucleotide excision repair (NER) of DNA. Particularly to exert the latter function, complex formation with the XPC protein, the pivotal NER damage recognition factor, is crucial. Here, we show that the C-terminal half of centrin-2, containing two calcium-binding EF-hand motifs, is necessary and sufficient for both its localization to the centrosome and interaction with XPC. In XPC-deficient cells, nuclear localization of overexpressed centrin-2 largely depends on co-overexpression of XPC, and mutational analyses of the C-terminal domain suggest that XPC and the major binding partner in the centrosome share a common binding surface on the centrin-2 molecule. On the other hand, the N-terminal domain of centrin-2 also contains two EF-hand motifs but shows only low-binding affinity for calcium ions. Although the N-terminal domain is dispensable for enhancement of the DNA damage recognition activity of XPC, it contributes to augmenting rather weak physical interaction between XPC and XPA, another key factor involved in NER. These results suggest that centrin-2 may have evolved to bridge two protein factors, one with high affinity and the other with low affinity, thereby allowing delicate regulation of various biological processes. PMID:23716636
A community-based study of nucleotide excision repair polymorphisms in relation to risk of non-melanoma skin cancer
Wheless, Lee; Kistner-Griffin, Emily; Jorgensen, Timothy J.; Ruczinski, Ingo; Berthier-Schaad, Yvette; Kessing, Bailey; Hoffman-Bolton, Judith; Francis, Lesley; Shugart, Yin Yao; Strickland, Paul T.; Kao, W.H. Linda; Alani, Rhoda M.; Smith, Michael W.; Alberg, Anthony J.
Nucleotide excision repair (NER) is responsible for protecting DNA in skin cells against ultraviolet radiation-induced damage. Using a candidate pathway approach, a matched case-control study nested within a prospective, community-based cohort was carried out to test the hypothesis that single nucleotide polymorphisms (SNPs) in NER genes are associated with susceptibility to non-melanoma skin cancer (NMSC). Histologically-confirmed cases of NMSC (n=900) were matched to controls (n=900) on age, gender, and skin type. Associations were measured between NMSC and 221 SNPs in 26 NER genes. Using the additive model, two tightly linked functional SNPs in ERCC6 were significantly associated with increased risk of NMSC: rs2228527 (odds ratio (OR) 1.57, 95% confidence interval (CI) 1.20 – 2.05), and rs2228529 (OR 1.57, 95% CI 1.20 – 2.05). These associations were confined to basal cell carcinoma of the skin (BCC) (rs2228529, OR 1.78, 95% CI 1.30 – 2.44; rs2228527 OR 1.78, 95% CI 1.31 – 2.43). These hypothesis-generating findings suggest functional variants in ERCC6 may be associated with an increased risk of NMSC that may be specific to BCC. PMID:22336945
C. elegans lifespan extension by osmotic stress requires FUdR, base excision repair, FOXO, and sirtuins.
Anderson, Edward N; Corkins, Mark E; Li, Jia-Cheng; Singh, Komudi; Parsons, Sadé; Tucey, Tim M; Sorkaç, Altar; Huang, Huiyan; Dimitriadi, Maria; Sinclair, David A; Hart, Anne C
Moderate stress can increase lifespan by hormesis, a beneficial low-level induction of stress response pathways. 5'-fluorodeoxyuridine (FUdR) is commonly used to sterilize Caenorhabditis elegans in aging experiments. However, FUdR alters lifespan in some genotypes and induces resistance to thermal and proteotoxic stress. We report that hypertonic stress in combination with FUdR treatment or inhibition of the FUdR target thymidylate synthase, TYMS-1, extends C. elegans lifespan by up to 30%. By contrast, in the absence of FUdR, hypertonic stress decreases lifespan. Adaptation to hypertonic stress requires diminished Notch signaling and loss of Notch co-ligands leads to lifespan extension only in combination with FUdR. Either FUdR treatment or TYMS-1 loss induced resistance to acute hypertonic stress, anoxia, and thermal stress. FUdR treatment increased expression of DAF-16 FOXO and the osmolyte biosynthesis enzyme GPDH-1. FUdR-induced hypertonic stress resistance was partially dependent on sirtuins and base excision repair (BER) pathways, while FUdR-induced lifespan extension under hypertonic stress conditions requires DAF-16, BER, and sirtuin function. Combined, these results demonstrate that FUdR, through inhibition of TYMS-1, activates stress response pathways in somatic tissues to confer hormetic resistance to acute and chronic stress. C. elegans lifespan studies using FUdR may need re-interpretation in light of this work. PMID:26854551
A mechanism for the exclusion of low-fidelity human Y-family DNA polymerases from base excision repair.
Haracska, Lajos; Prakash, Louise; Prakash, Satya
The human Y-family DNA polymerases, Poliota, Poleta, and Polkappa, function in promoting replication through DNA lesions. However, because of their low fidelity, any involvement of these polymerases in DNA synthesis during base excision repair (BER) would be highly mutagenic. Mechanisms, therefore, must exist to exclude their participation in BER. Here, we show that although Poliota, Poleta, and Polkappa are all able to form a covalent Schiff base intermediate with the 5'-deoxyribose phosphate (5'-dRP) residue that results from the incision of DNA at an abasic site by an AP endonuclease, they all lack the ability for the subsequent catalytic removal of the 5'-dRP group. Instead, the covalent trapping of these polymerases by the 5'-dRP residue inhibits their DNA synthetic activity during BER. The unprecedented ability of these polymerases for robust Schiff base formation without the release of the 5'-dRP product provides a means of preventing their participation in the DNA synthetic step of BER, thereby avoiding the high incidence of mutagenesis and carcinogenesis that would otherwise occur. PMID:14630940
Role of the DNA Base Excision Repair Protein, APE1 in Cisplatin, Oxaliplatin, or Carboplatin Induced Sensory Neuropathy
Kelley, Mark R.; Jiang, Yanlin; Guo, Chunlu; Reed, April; Meng, Hongdi; Vasko, Michael R.
Although chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting side effect of platinum drugs, the mechanisms of this toxicity remain unknown. Previous work in our laboratory suggests that cisplatin-induced CIPN is secondary to DNA damage which is susceptible to base excision repair (BER). To further examine this hypothesis, we studied the effects of cisplatin, oxaliplatin, and carboplatin on cell survival, DNA damage, ROS production, and functional endpoints in rat sensory neurons in culture in the absence or presence of reduced expression of the BER protein AP endonuclease/redox factor-1 (APE1). Using an in situ model of peptidergic sensory neuron function, we examined the effects of the platinum drugs on hind limb capsaicin-evoked vasodilatation. Exposing sensory neurons in culture to the three platinum drugs caused a concentration-dependent increase in apoptosis and cell death, although the concentrations of carboplatin were 10 fold higher than cisplatin. As previously observed with cisplatin, oxaliplatin and carboplatin also increased DNA damage as indicated by an increase in phospho-H2AX and reduced the capsaicin-evoked release of CGRP from neuronal cultures. Both cisplatin and oxaliplatin increased the production of ROS as well as 8-oxoguanine DNA adduct levels, whereas carboplatin did not. Reducing levels of APE1 in neuronal cultures augmented the cisplatin and oxaliplatin induced toxicity, but did not alter the effects of carboplatin. Using an in vivo model, systemic injection of cisplatin (3 mg/kg), oxaliplatin (3 mg/kg), or carboplatin (30 mg/kg) once a week for three weeks caused a decrease in capsaicin-evoked vasodilatation, which was delayed in onset. The effects of cisplatin on capsaicin-evoked vasodilatation were attenuated by chronic administration of E3330, a redox inhibitor of APE1 that serendipitously enhances APE1 DNA repair activity in sensory neurons. These outcomes support the importance of the BER pathway, and particularly APE
A ubiquitylation site in Cockayne syndrome B required for repair of oxidative DNA damage, but not for transcription-coupled nucleotide excision repair.
Ranes, Michael; Boeing, Stefan; Wang, Yuming; Wienholz, Franziska; Menoni, Hervé; Walker, Jane; Encheva, Vesela; Chakravarty, Probir; Mari, Pierre-Olivier; Stewart, Aengus; Giglia-Mari, Giuseppina; Snijders, Ambrosius P; Vermeulen, Wim; Svejstrup, Jesper Q
Cockayne syndrome B (CSB), best known for its role in transcription-coupled nucleotide excision repair (TC-NER), contains a ubiquitin-binding domain (UBD), but the functional connection between protein ubiquitylation and this UBD remains unclear. Here, we show that CSB is regulated via site-specific ubiquitylation. Mass spectrometry analysis of CSB identified lysine (K) 991 as a ubiquitylation site. Intriguingly, mutation of this residue (K991R) does not affect CSB's catalytic activity or protein stability, but greatly affects genome stability, even in the absence of induced DNA damage. Moreover, cells expressing CSB K991R are sensitive to oxidative DNA damage, but proficient for TC-NER. K991 becomes ubiquitylated upon oxidative DNA damage, and while CSB K991R is recruited normally to such damage, it fails to dissociate in a timely manner, suggesting a requirement for K991 ubiquitylation in CSB activation. Interestingly, deletion of CSB's UBD gives rise to oxidative damage sensitivity as well, while CSB ΔUBD and CSB K991R affects expression of overlapping groups of genes, further indicating a functional connection. Together, these results shed new light on the regulation of CSB, with K991R representing an important separation-of-function-mutation in this multi-functional protein. PMID:27060134
A ubiquitylation site in Cockayne syndrome B required for repair of oxidative DNA damage, but not for transcription-coupled nucleotide excision repair
Ranes, Michael; Boeing, Stefan; Wang, Yuming; Wienholz, Franziska; Menoni, Hervé; Walker, Jane; Encheva, Vesela; Chakravarty, Probir; Mari, Pierre-Olivier; Stewart, Aengus; Giglia-Mari, Giuseppina; Snijders, Ambrosius P.; Vermeulen, Wim; Svejstrup, Jesper Q.
Cockayne syndrome B (CSB), best known for its role in transcription-coupled nucleotide excision repair (TC-NER), contains a ubiquitin-binding domain (UBD), but the functional connection between protein ubiquitylation and this UBD remains unclear. Here, we show that CSB is regulated via site-specific ubiquitylation. Mass spectrometry analysis of CSB identified lysine (K) 991 as a ubiquitylation site. Intriguingly, mutation of this residue (K991R) does not affect CSB's catalytic activity or protein stability, but greatly affects genome stability, even in the absence of induced DNA damage. Moreover, cells expressing CSB K991R are sensitive to oxidative DNA damage, but proficient for TC-NER. K991 becomes ubiquitylated upon oxidative DNA damage, and while CSB K991R is recruited normally to such damage, it fails to dissociate in a timely manner, suggesting a requirement for K991 ubiquitylation in CSB activation. Interestingly, deletion of CSB's UBD gives rise to oxidative damage sensitivity as well, while CSB ΔUBD and CSB K991R affects expression of overlapping groups of genes, further indicating a functional connection. Together, these results shed new light on the regulation of CSB, with K991R representing an important separation-of-function-mutation in this multi-functional protein. PMID:27060134