the integrity of dna is an important aspect of cell survival , since the molecule carries hereditary information and instructs essential biological processes such as transcription and replication of living cells . the various cancer drugs that have been used in chemotherapy over the last 60 years kill cells in different ways . in addition to the targeted therapies developed over the last two decades , many routinely used anticancer agents ( topoisomerase i / ii inhibitors , dna alkylating agents , and antimetabolites ) target the dna helix itself . watson and crick 's discovery of the dna double helix in 1953   led to extensive research in the field of the interactions between small molecules ( whether of natural or synthetic origin ) with nucleic acids . in turn , this work prompted the widespread use of some of these molecules as anticancer agents [ 37 ] . the interaction between small ligands and dna involves either ( i ) nonspecific binding through electrostatic interactions with the negatively charged sugar - phosphate backbone , ( ii ) intercalation of the ligand 's planar aromatic rings between two adjacent base pairs ( see figure 1 ) , or ( iii ) major- or minor - groove binding . following dna recognition by anticancer compounds , the subsequent interaction can either be noncovalent ( dna ligands ) or covalent ( alkylating agents ) . whereas most dna - interacting compounds stabilize the dna double helix , a few display the particular ability to destabilize it  leading potentially to various cellular consequences . the dna double helix is conventionally illustrated as a spiral staircase , in which the two strands ( the handrails ) are stabilized by hydrogen bonds between the watson - crick base pairs ( the steps ) . however , these  steps  are not stable because their noncovalent interactions are reversible . depending on the dna sequence , denaturation ( melting ) can be local or widespread [ 8 , 9 ] and enables various crucial cellular processes ( including dna replication , transcription , and repair ) to take place [ 1012 ] . both sequence specificity and interaction ( whether covalent or not ) with a small compound or a protein can induce tilt , roll , and twist effects ( a rotation of the base pairs in the x , y , or z axis , respectively  figure 1 ) and therefore change the helix 's overall organization . furthermore , slide or flip effects can also modify the geometrical orientation of the dna helix ( figure 1 ) . hence , the flip effect and ( to a lesser extent ) the other above - defined movements modulate the double - strand stability within the helix or at its ends . indeed , under physiological conditions , local dna  breathing has been evidenced at both ends of the dna helix   and b - to - z dna structural transitions have been observed in internal dna regions   in a sequence - dependent manner [ 8 , 1629 ] . these types of locally open dna structures are good substrates for specific proteins ( such as single - strand binding proteins , ssbps ) which can also induce the opening of a  closed  dna helix . in addition to naturally occurring dna breathing , the helix can also be unzipped by cellular proteins and dna binding compounds ( some of which are used in the clinic ) . in order to achieve essential cellular processes such as dna transcription , replication and repair , some cellular proteins are able to naturally unzip the dna helix . the most well known of these ( dna helicases ) are essential players in the above - mentioned processes . the destabilization is obtained through either an active , direct separation of the two dna strands [ 3133 ] or a passive opening mode in which the helicase binds to the locally single - stranded dna portion generated by base pairing fluctuation ( which mostly depends on the dna sequence and induces prebent dna structures ) [ 3436 ] . after dna opening , the helicase partially translocates to the generated single - stranded dna regions and subsequently moves along the base pairs to unwind the double helix at up to 5001000  bps . the blm helicase ( the human recq helicase responsible for bloom 's syndrome , which is characterized by dna repair deficiencies ) actively destabilizes the dna duplex and performs rapid , efficient dna strand separation . helicases are not the only proteins with intrinsic double - strand dna opening ability ; this is also a property of replication protein a ( rpa ) , a very efficient dna destabilizing protein involved in many dna metabolism processes ( including repair , replication , and recombination ) [ 3941 ] . rpa is a mammalian nuclear ssbp ; the ssbp family members ( comprising eukaryotic , bacterial and viral proteins ) can efficiently destabilize dna helix by unwinding up to one thousand base pairs . similarly , the mouse myeloma helix - destabilizing protein , the calf thymus hnrnp - related protein up1 and the mammalian p8 protein ( related to glyceraldehyde-3-phosphate - dehydrogenase ) also present both dna single - strand binding and dna helix - destabilizing abilities , as evidenced in thermal denaturation measurements [ 4244 ] . high - mobility group ( hmg ) proteins are structurally and functionally important chromatin components which also display dna destabilizing activities . indeed , melting studies have revealed that both hmg1 and hmg2 destabilize dna in the presence of 25 to 100  mm nacl but stabilize dna in the absence of salt [ 45 , 46 ] . an hmg - related dna binding domain with dna - destabilization properties has been found within c - abl kinase protein . moreover , this dna destabilization was shown to increase the extent of hmg protein binding to dna in the vicinity of the c - abl binding site [ 47 , 48 ] . more recently , it has been reported that the nucleocapsid protein of hiv-1 can destabilize dna   via its dna - bending activity . more specifically , the dna - destabilization function involves the protein 's first zinc finger , bearing residues ile24 and asn27 . the latter 's pathological mechanism of action involves translocation to the nucleus , where the protein binds chromatin and converts to insoluble aggregates . using fret - coupled dna - melting temperature studies , prion protein was found to induce significant dna bending , unwinding , and thus local destabilization of the dna helix . although the above - mentioned proteins induce relatively large dna destabilization effects , small modifications ( such as base flipping ) could also perturb the local stability of dna . various dna nucleotide excision repair ( ner ) proteins [ 54 , 55 ] , base excision repair proteins [ 5661 ] , and dna methyltransferases [ 62 , 63 ] ( such as cytosine - c5-methyltransferase ) are known to promote base flipping . more recently , base flipping has been described in the recognition of methylated bases by the set and ring - associated ( sra ) domain protein uhrf-1 [ 65 , 66 ] and dna binding by the transcription factor nf-b . small compounds that interact noncovalently with dna can bind to the minor or major groove between the two walls of the dna helix , via intercalation between two planar  rungs  of the base pase staircase ( figure 1 ) or covalently as a result of dna alkylation . most of the dna unwinding compounds with well - defined binding modes belong to the intercalating or alkylating groups . mono- and bis - intercalators present their intercalative rings between adjacent base pairs in parallel or perpendicular ways . this results in ( i ) unwinding of the dna helix by an angle x , where x < 36 ( since 36 is the rotation angle between two adjacent base pairs in native dna ) , and ( ii ) subsequent elongation of the dna ( length ) . the value of x depends on the nature of the interacting compound ( the rotated orange arrow in figure 1 ) .   with dna alkylating agents , dna destabilization can arise from dna bending , base flipping , or much more extensive dna opening ( figure 1 ) . dna - intercalating agents which impair the stability of the helix can be either mono- or bis - intercalators . acridine orange ( ao ) ( figure 2 ) is well known for its ability to intercalate between double - stranded dna but can also bind single - stranded dna with high affinity . when bound to dna , ao fluoresces at different wavelengths , depending on the nature of the nucleic acid ; green fluorescence occurs after binding to double - strand dna , whereas red luminescence results from interaction with single - strand dna . thermal denaturation studies suggest that the overall stability of the dna double helix is increased by ao binding . however , local distortion and denaturation of double - stranded dna are also generated , as evidenced by formaldehyde and diethylpyrocarbonate ( depc ) probing . dna denaturation after ao binding was also confirmed by spectral and thermodynamic data   and in situ experiments [ 71 , 72 ] . ellipticine and adriamycin ( figure 2 ) also induce local unzipping of the dna helix ; the dna melting temperature ( tm ) falls by 5.1 and 4.8c , respectively , [ 69 , 70 , 75 ] . as with ao , these two compounds bind efficiently to single - stranded dna [ 76 , 77 ] . this contrasts with ethidium bromide 's binding to nucleic acids , which is highly specific for double - stranded dna and does not destabilize the double helix . ellipticine , adriamycin , and ao all intercalate between two adjacent base pairs and then subsequently change their orientation to interact with the single - stranded nucleic acid sections formed locally during dna breathing . the single - stranded portions within the dna helix lengthen because of cooperative binding by the intercalator , which thus leads to a higher level of dna denaturation . this progressive unzipping of the dna helix has also been observed in situ using cytometry , with a direct relationship between the decrease in green fluorescence and increased concentrations of ao in treated cells or nuclei . furthermore , post denaturation aggregate formation was observed using electron microscopy , with dna condensation occurring primarily in heterochromatin , ribosomal , and polysomal structures [ 71 , 72 ] . within the bis - intercalating group of compounds , the cyclo - bis - intercalator bisacridine a ( bisa ) ( figure 2 ) also displayed dna unwinding properties . this macrocyclic compound is composed of two acridine cores ( the dna - intercalating motifs ) linked by polyammonium bridges . by using a variety of complementary biochemical and biophysical techniques ( such as fluorescence , melting temperature studies , and gel electrophoresis ) , slama - schwok et al . nicely demonstrated the ability of the polyaminomacrocycle bisa to shift the equilibrium from duplex dna towards hairpin nucleic acid structures   and to destabilize double - stranded dna   ( two properties not observed with monoacridine derivatives ) . additionally , when irradiated with light , bisa efficiently induces photocleavage through its acridine photoactive core . this activity is greater with single - stranded nucleic acids than with double - stranded nucleic acids . interestingly , nmr and molecular modelling studies of bisa compounds bound to an abasic site - containing dna show that one acridine ring intercalates between the ca and tg base pairs , the second ring lies in the free space of an at base pairing and the linker chains are positioned in the major and minor grooves on each side . on the base - pair level , tg mismatches and apt recognition result in base flipping of the thymine  suggesting that bisa sterically prevents dna glycosylases from binding to their specific , base - damaged recognition sites . however , some covalent dna - binding anticancer drugs can also locally destabilize the dna helix ; these include the well - known alkylating agent cisplatin and its derivatives and the recent drug candidate s23906 - 1 ( a benzo - acronycine derivative ) . this contrasts with the dna stabilization properties of most dna - alkylating agents ( whether used in chemotherapy or not ) , such as mitomycin c , some psoralen and dinuclear platinum derivatives , ecteinascidine 743 and nitrogen mustards [ 8591 ] . cisplatin ( cis - diaminedichloridoplatinum(ii ) ; figure 3 ) was one of the first chemotherapeutic agents to be developed and is still frequently used in the clinic . it is able to form inter- and intrastrand crosslinks and monovalent adducts , primarily though covalent bonding to the n7 atom of guanine residues . the most common lesion is the intra - strand crosslink ( occurring preferentially ( 65% ) at the gpg dinucleotide ) , followed by apg intrastrand crosslinks ( 25% ) . interstrand crosslinks occur less frequently and depending on the nature of the platinated agent , with 5 to 8% for cisplatin , 12% for transplatin ( figure 3 ) and up to 30% for trans - ptcl2(nh3)(quinoline ) ( figure 3 ) and trans - ptcl2(nh3)(thiazole ) derivatives . in comparison , nitrogen mustards induce 1 to 5% of inter - strand crosslinks , whereas nitrosourea and mitomycin c induce 2 to 8% and 5 to 13% , respectively , [ 9395 ] . cisplatin - induced intra - strand crosslinks at gpg base pairs result in ( i ) bending of the dna axis toward the major groove with an angle of 5578 , and ( ii ) dna distortion , enabling local denaturation of the double helix via destabilization of the watson - crick base pairing [ 96102 ] . in comparison , the bending angle for inter - strand crosslinks is 45 and is associated with dna unwinding of 79  4. the distortion in platinated - gpg intra - strand crosslinks is different and depends on the sequence context , with up to 7  bp for 1,3-intrastrand crosslinks in a tgtgt context . this destabilization was found to be enthalpic ( rather than entropic ) in origin [ 101 , 103 ] . similarly , cisplatin adducts occurring at the 5-tggt sequence induce a decrease of more than 10c in the melting temperature  much higher than the decrease of 6c or so measured for 5-cggt and 5-aggc sequences [ 104 , 105 ] . in contrast to cisplatin , the interstrand crosslinks formed by transplatin do not destabilize the dna helix or correlate with changes in the transition entropy or enthalpy [ 101 , 107 ] . interestingly , the third - generation platinum antitumor derivative oxaliplatin [ ( 1r,2r - diamminocyclohexane)-oxalatoplatinum-(ii ) ] ( figure 3 ) was found to induce greater dna unwinding , bending and helix destabilization than cisplatin . this correlates with the lower degree of cellular dna damage seen after oxaliplatin treatment than after cisplatin treatment and the lower hmg protein affinity for oxaliplatin - versus cisplatin - induced damage ; these findings suggest that the two molecules induce different dna repair processes / efficiencies , depending on the extent of local helix destabilization . it is noteworthy that some other bifunctional platinated derivatives do not destabilize the dna duplex . this is the case for pyrazolato - bridged dinuclear platinum(ii ) complex [ ( cis-{pt(nh3)2})2(mu - oh)(mu - pyrazolate ) ] , which crosslinks two adjacent guanines and unwinds the dna by around 15 but does not change the directionality of the helix axis . the dna sequence is also important ; monofunctional platinum adducts exhibit different dna destabilizing effects depending on the base sequences surrounding the guanine target site [ 97 , 109 ] . indeed , when oligonucleotide containing platinum adducts were incubated in the presence of 50 or 500  mm nacl , the highest dna destabilization was observed when the guanine target was located within the tgc triplet sequence ( with a tm value of 10.6c and 13.2c , resp . ) . for all triplets , the decrease in tm was greater in 500  mm nacl buffer than in 50  mm na counterion containing buffer . in general , the highest dna destabilization effect was seen when the monoadduct was positioned between pyrimidine residues . osmium tetroxide ( oso4 ) and depc probing revealed that both thymine and the opposite adenine are crucial for the local distortion of the dna structure by the platinum mono - adduct positioned within a 5-tgc triplet but not within a 5-agt or 5-tga triplet . in contrast , none of these chemical probes reacted with the bifunctional adduct at the 5-tggt sequence . found that the cisplatin derivatives cis-[ptcl(nh3)2(oh2 ) ] , cis-[ptcl(nh3)(c - c6h11nh2)(oh2 ) ] , and trans-[ptcl(nh3)(quinoline)(oh2)]destabilized both double - stranded dna and double - stranded rna ( figure 3 ) . indeed , after platination with these compounds , the melting temperatures for both the rna and dna hairpins fell . with hairpin rna , platination induced much weaker destabilization , with a tm of 5c . in the case of dna , the platinum - induced destabilization was more pronounced , with tm values of around 11c . this is the case for ruthenium derivatives such as [ ( -p - cymene)ru(ii)(en)-(cl ) ] ( ru - cym , figure 4 ) . this organometallic ruthenium(ii ) arene complex was rationally designed on the basis that changing the metal ion from platinum to ruthenium should provide additional coordination sites in the octahedral complexes , modify the oxidation rate , and change the ligand affinity and binding kinetics for use in chemotherapy [ 7 , 112114 ] . in particular , brabec and co - workers performed tm studies while varying the drug / dna ratio in buffer containing naclo4 concentrations ranging from 0.01  m to 0.2  m [ 115 , 116 ] . tm measurements at a drug / dna ratio of 0.1 showed a decrease of up to 4c in the ct - dna melting temperature at all na concentrations . this dna helix destabilization was also observed using biphenyl ( ru - bip ) , dihydroanthracene ( ru - dha ) , and tetrahydroanthracene ( ru - tha ) ( figure 4 ) as arenes but only at the highest concentration of naclo4 ; at lower na - counterion concentrations , ru - bip , ru - dha and ru - tha induced dna helix stabilization , due to a positive charge effect on the ruthenium moiety and the intercalation process . when compared with the other ruthenium arene complexes , the dna helix destabilization activity of ru - cym correlates with a smaller unwinding angle of 7 ( versus 14 of unwinding in supercoiled plasmid dna by ru - bip , ru - dha and ru - tha ) . the ru - cym - induced dna - unwinding appears to be consistent with the absence of intercalation of ru - cym between two adjacent base pairs and the formation of monoadducts on the n7 atom of guanine residues . ru - tha and ru - cym have been used as models for the repair of dna - ruthenium complexes the compounds destabilize the dna helix via different enthalpic effects and differ in terms of their dna base - pair intercalation propensity . dna destabilization was also recently evidenced for new ruthenium derivatives , such as monodentate - ru(ii ) . in terms of dna stabilization / destabilization properties , the psoralen derivative 4-(hydroxymethyl)-4 , 5,8-trimethylpsoralen ( hmt ) ( figure 5 ) exerts two effects : ( i ) monoaddition of a psoralen residue stabilizes the double helix formed by two non - self - complementary oligonucleotides by as much as 1.3  kcal / mol ( for a furan - side mono - adduct ) or 0.7  kcal / mol ( for a pyrone - side mono - adduct ) at 25c in 50  mm nacl ; ( ii ) mono - addition of a psoralen residue to each of the two thymidines in the double helix in the sequence gggtaccc destabilizes the helix by 1.8  kcal / mol at 25c in 1 m nacl  the two hmt molecules at the centre of each strand cause an unfavourable enthalpy change and a favourable entropy change . in the cell , the environmental and tobacco smoke carcinogen benzo[a]pyrene ( bap ) is metabolized into ( + /)-anti - benzo [ a]pyrene-7,8-dihydrodiol-9,10-epoxide ( bpde ) ( figure 5 ) . the ( + ) -7r,8s,9s,10r enantiomer ( + ) -anti - bpde is thought to be the metabolite that is ultimately responsible for mutations , dna damage , and cancer . by covalently linking to the exocyclic nh2 group of guanine , bpde forms a bulky dna adduct in the minor groove of the helix and destabilizes base pairing [ 120 , 121 ] . several enantiomers are produced by the alkylation reaction which follows the opening of the epoxide group . the ( + ) -anti - bpde adducts consist mostly of the carcinogenic 10s ( + ) -trans - anti - bpde ( derived from the ( + ) -7r,8s,9s,10r compound ) and , to a lesser extent , stereoisomeric 10r ( + ) -cis - b [ a]p - n2-dg adducts . the ( )-bpde enantiomer forms ( )-trans - b [ a]p - n2-dg adducts but with lower efficiency . the dna bonding of the stereoisomeric damage suggests base displaced intercalation or minor groove conformations . covalent adduct formation prevents the amino group of guanine from hydrogen bonding with the opposite cytosine ( which otherwise stabilizes gc base pairs in the native dna helix ) . this results in base flipping , with the ( + ) -anti - b [ a]p - n2-dg bulky adduct on the guanine situated in the minor groove and the opposite cytosine aligned with the major groove [ 122 , 123 ] . depending on the target sequence , the bulky 10s ( + ) -trans - anti - b [ a]p - n2-dg rings point in the 5 direction relative to the alkylated guanine position in each case , although the exact positions are different . indeed , using 5-cgg*c dna , the 10s ( + ) -trans - anti - b [ a]p - n2-dg lesion untwists the dna significantly and causes a large bend in the dna helix . in contrast , with a 5-cggc sequence , no untwisting is seen but the dna helix is destabilized 5 to the lesion . these structural differences result in differing electrophoretic mobility in polyacrylamide gels and different protein / dna recognition and dna repair efficiencies . although the baps are environmental mutagens and not antitumor agents , their very particular mode of dna binding with dual interference on dna repair processes could highlight useful phenomena involved in the mechanism of action of cancer drugs ( such as effects on dna repair , as described in section 5 ) . as is the case for bap , 4-ohen compounds are derived from equine oestrogens ( equilin , 3-hydroxy-1,3,5(10),7-estratetren-17-one , and equilenin , 3-hydroxy-1,3,5(10),6,8-estrapenten-17-one ) which are present at various concentrations in the hormone substitution therapies used to reduce the side effects of the menopause but which are also thought to contribute to a greater risk of breast cancer in the treated population [ 126128 ] . in the body , both equilin and equilenin are rapidly converted into the intermediate catechol 4-hydroxyequilenin , which is further oxidized into the reactive 4-hydroxyequilenin - o - quinone ( figure 5 ) . this ortho - quinone form of 4-ohen is a potent cytotoxic and genotoxic agent   and forms a bulky lesion on da , dc , and dg but not t residues [ 131134 ] . this damage can be detected not only in cell culture but also in breast cancer biopsies from patients having undergone hormone substitution therapy . each of the base adducts are present as four stereoisomers , each of which induces different levels of structural distortion in duplex dna [ 136138 ] . 4-ohen - c adducts present an unusual cyclic core with the bulky rings pointing along the major or the minor groove depending on whether the glycosidic bond adopts a syn- or anticonformation , respectively , . alkylation of an 11-bp oligonucleotide at specific da or dc residues results in a strong decrease in the melting temperature of the double - stranded dna , compared with the unmodified oligonucleotide . the magnitude of the decrease depends on the position of the adduct within the oligonucleotide : a 69c decrease in tm is obtained when the adduct is located 1 or 2-bp from the end of the 11-bp dna , whereas a large ( 2127c ) decrease is induced when the adduct is present more centrally ( positions 4 to 8) . the extent of the destabilization also depends on the adduct 's stereoisomeric orientation , as defined using circular dichroism measurements , thermodynamic analyses and molecular modelling . indeed , distortions , base - stacking characteristics , and groove sizes were found to vary according to the nature and the stereoisomerism of the bulky dna lesion . the compound s23906 - 1 [ ( + ) -cis-1,2-diacetoxy-6-methoxy-3,3,14-trimethyl-1,2,3 - 14-tetrahydro-7h- benzo[b]pyrano[3,2-h]acridin-7-one ] ( figure 5 ) is a potent dna - alkylating agent with strong cytotoxic and antitumour properties . on the basis of very promising preclinical trials s23906 - 1 alkylates dna on guanine 's exocyclic amino group ( located in the minor groove of the dna helix ) and thus contrasts with commonly used chemotherapeutic alkylating agents which react at guanine 's n7 position in the major groove ( cisplatin , nitrosourea , nitrogen mustards , etc . ) . this nucleophilic point is also targeted by other clinically used antitumour agents , such as ecteinascidine-743 ( et-743/trabectedin / yondelis ) developed by the company pharmamar ( spain ) , mitomycin - c ( a dual alkylating agent which bonds to either the n2 group of guanine in the minor groove or the n7 group of guanine in the major groove )   and anthramycin . in contrast to et-743 , mitomycin - c and the anthramycins , the alkylation of double - stranded dna by s23906 - 1 results in local destabilization of the dna helix and thus the formation of a single - stranded dna portion that can be attacked by single - strand - specific nucleases ( such as nuclease s1 ) . this destabilization was seen with various multiple derivatives of s23906 - 1 ( e.g. , esters of the benzo [ b]acronycine core , figure 5 ) . the dna destabilization potency of this series was confirmed in a variety of physical and biochemical approaches . for example , quantification of the ratio between the fluorescence of picogreen ( a dye which interacts with both double- and single - stranded dna ) and ethidium bromide ( bet ) ( a double - stranded - specific dye ) revealed additional picogreen binding and suggested that s29385 - 1 generates single - strand dna ( figure 6(a ) ) . accordingly , dna melting temperature studies evidenced a negative tm value , reflecting dna destabilization ( figure 6(b ) ) . s23906 - 1 's ability to destabilize dna was also clearly demonstrated in biochemical approaches , such as electrophoretic mobility shift assays ( generating single - stranded dna form following alkylation of a fully double - stranded dna fragment ) and the use of nuclease - s1 single - strand - specific digestion to map the relative positions of locally dna openings induced by alkylation ( figure 7 and ) . compounds which change the equilibrium between the stable , double - stranded dna helix and locally destabilized strands could strongly alter protein / dna recognition and thus have major cellular consequences . indeed , base kinking , unstacking , and nucleotide extrusion ( flipping ) induce discontinuities in the double helix and thus facilitate dna lesion / mismatch recognition [ 143145 ] . the few literature studies to have addressed this point reveal that proteins which recognize damaged dna are affected by the stabilization or destabilization of the dna helix ; this leads to a major impact on biological parameters such as antitumour activity , transcription factor binding potency , and dna repair process efficiency [ 146148 ] . this type of cellular impact has been particularly well demonstrated for the platinated adducts recognized by hmg proteins . hmg / platinated dna recognition is triggered by the strong dna bending generated by the platinated agent ( figure 2 ) . the large induced bend fits perfectly with the l - shaped structure of hmg dna binding domain ( hmg - box ) and reduces the  cost  of dna bending for the protein . as a consequence , hmg proteins bind less well to oxaliplatin adducts than to cisplatin adducts because the former agent induces relatively greater dna bending and thus stronger dna destabilization . this finding correlates with the lower level of dna lesions found in cells treated with oxaliplatin , relative to cisplatin . it is assumed that hmg binding shields the platinated adducts from repair by the human dna excision machinery   and therefore participates in platinated - agent - induced cytotoxicity . however , too strong a bend and greater dna destabilization lead to a structure that does not fit perfectly with l - shaped hmg - box , thus resulting in a weaker hmg protein binding . the weaker hmg binding to oxaliplatin adducts corresponds to weaker protection from dna repair and so oxaliplatin - dna lesions are more efficiently repaired than cisplatin - induced lesions . this results in a lower number of lesions in cells for oxaliplatin than for cisplatin . moreover , bent platinated - dna is a good substrate for transcription factors such as sry and lef-1 ( which belong to the hmg - box family , regarding their dna - binding domain ) and explains the observation of transcriptional changes in treated cells [ 92 , 154156 ] . regarding dna repair , the local destabilization of the double helix , base flipping and poor base stacking all play a role in the recognition of dna lesions by repair proteins [ 157160 ] . this has been well demonstrated for the msh2/msh6 heterodimer mismatch repair complex ( mut - s alpha ) , which recognizes not only mismatched bases but also certain dna lesions , such as cisplatin ( but not transplatin ) crosslinks [ 161165 ] .   in the case of ruthenium - derivative - induced dna lesions , the ner machinery appears to be less efficient than for platinum adducts . interestingly , ru - cym adducts ( which destabilize the dna helix much more than ru - tha adducts ) are excised more efficiently than ru - tha complex adducts . this is consistent with the lower binding by rpa to dna containing ru - tha adducts and ( to a lesser extent ) ru - cym / dna damage . the observation that ru - tha was much more cytotoxic than ru - cym in both a2780 human ovarian cancer cells and the ht29 colon carcinoma cell line suggests that dna intercalation has a major role in the cytotoxicity of these dna - destabilizing derivatives . in prokaryotes , the ner sensor protein uvrb efficiently recognizes bap lesions through lesion - induced local thermodynamic distortion / destabilization and nucleotide flipping . some variations in the excision efficiency ( up to a factor of 3-fold ) are observed , depending on the stereoisomeric orientation of the dna adducts ( i.e. , ( + ) or (  ) , cis- or trans- ) . in eukaryotes , the bap lesions are usually recognized by the ner machinery 's  sensor  protein xpc , which then initiates dna repair in association with the hr23b protein [ 168 , 169 ] . in particular , it has been suggested that xpc / hr23b 's weaker recognition of ( + ) -trans - b [ a]p - n2-dg adducts ( relative to the other conformers ) contributes to its higher mutagenic and tumorigenic activity . xpc binding requires dna bending   and is facilitated by local conformational flexibility [ 143 , 144 , 171 ] and destabilization of the base pairing , as evidenced by several model dna lesions ( such as thymine - glycol ) . on the cellular level , human bronchial epithelial 16hbe cells treated with bap ( as a source of reactive bpde ) displayed greater expression of the ner proteins xpa and xpg and the heat shock protein hsp70 . subcellular analysis with confocal microscopy evidenced nuclear colocalization of hsp70 with xpa and xpg after bap treatment , suggesting that hsp70 has a role in the cellular dna repair response . accordingly , ( + /)-anti - bpde induces chromosome instability and centromere amplification in lung cells . the cellular consequences of ( + /)-anti - bpde treatment were also assessed using a whole - genome microarray technique in normal human amnion epithelial cells ; the researchers observed downregulation of the expression of genes involved in signal transduction , cytoskeleton , dna repair , metabolism and regulation of transcription and the cell cycle , with features similar to those observed after cell irradiation with uv - light [ 175 , 176 ] . it was recently reported that the structural differences observed for an identical , highly mutagenic ,   ( + ) -(7r,8s,9s,10r)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo [ a]pyrene - dna lesion lead to different repair processes as a function to the sequence contexts . indeed , in cell - free human hela extracts , destabilized dna at a 5-cg*gc site was more rapidly excised than the bent dna at a 5-cgg*c site . since the dna helix is already opened up by alkylation , the dna repair protein recognition step ( including induced base flipping ) requires less energy and thus is potentially more rapid for dna that has already been destabilized ( at a 5-cggc site ) than for bent duplex dna ( the 5-cggc site ) . this study clearly emphasized the importance of the dna sequence context for efficient adduct repair . isomer - dependant dna repair potency is also assumed to occur with bulky catechol 4-ohen - adducts , which ner proteins excise with an efficiency that depends on the alkylated base , the stereoisomerism of the adducts and the sequence context . for example , 4-ohen - dc adducts are more efficiently excised than 4-ohen - da adducts . interestingly , it was shown in male zebrafish that 17a - ethinylestradiol ( a source of 4-ohen ) is able to decrease ner efficiency and the expression of ner genes such as xpc , xpa , xpd , and xpf ( but not hr23b ) [ 179 , 180 ] . in the search for new cancer drugs with novel mechanisms of action and on the basis of promising preclinical testing , the benzoacronycine derivative s23906 - 1 has entered phase i clinical trials as a racemate of two cis - diacetylated - enantiomers . as mentioned above , s23906 - 1 alkylates dna in the minor groove and induces strong destabilization of the double helix . given the presence of two reactive acetate groups on asymmetric carbons , two pairs of enantiomers can be formed : two cis ( 1r ; 2r and 1s ; 2s ) and two trans ( 1r ; 2s and 1s ; 2r ) structures . hence , s23906 - 1 is a mixture of 1r ; 2r and 1s ; 2s . we tested the ability of each of the pure cis - enantiomers not only to react with dna but also to destabilize the dna helix and thus affect single - stranded endonuclease activity . our results showed that dna destabilization depends on the orientation of the adduct core in the open drug / dna complex and correlates with differing cellular and antitumour effects : the enantiomer with the greatest dna destabilization presents the highest antitumour activity in animal models . little is known about the repair of s23906 - 1 dna adducts : the involvement of the ner proteins xpc and csb was recently found to be related to cell sensitivity to s23906 - 1 , associated with both global genome repair and transcription coupled ner . ongoing work is seeking to identify the proteins involved in s23906 - 1/dna adduct recognition and evaluate their impact on the compound 's cytotoxic activity . on one hand , locally destabilized dna could favour the recognition of a dna lesion by the dna repair  sensor  proteins , leading to an increase in the excision efficiency or rate . full repair after excision thus results in weaker antitumor activity , unless the dna repair process is blocked  as has been evidenced for the antitumour activity of et-743 ( yondelis ) . in this latter case , the et-743/dna adduct traps the xpg endonuclease protein involved in the ner machinery and increases the number of single - strand breaks . on the other hand , the wide , local opening of the dna helix prompted by this particular compound may increase cleavage by single - strand - specific nuclear endonucleases ; the greater number of double - strand breaks may then overwhelm the cancer cells ' dna repair capacity . drug - induced destabilization of the dna helix appears to be part of a novel antitumour mechanism of action and is associated with particular intercalation processes or postalkylation dna distortions . dna - destabilizing compounds are relatively rare and represent just a few drops in an ocean of dna - interacting molecules ( which primarily stabilize the double helix ) . the molecular and cellular consequences of this original binding mode differ from those induced by dna - stabilizing compounds . the dna replication machinery may also be affected because dna opening requires less energy when the double helix is already destabilized by a drug . in view of the little available literature data furthermore , we believe that it is important not to consider dna destabilization as a unique process . this phenomenon must be considered in relation to ( i ) the potentially associated bend in the dna helix ( as evidenced by the comparison between oxaliplatin- and cisplatin - induced distortions of dna   or the effect of the different isomers of bpde [ 125 , 177 ] ) , and ( ii ) the length of the locally destabilized dna , which varies according to the compound 's nature ( i.e. , the dna opening induced by benzoacronycine derivatives appears to be extensive enough to be susceptible to single - strand - specific nucleases , whereas other modifications are not ) . recent and ongoing studies of the impact of dna destabilization on dna repair and cytotoxicity activities illustrate the increasing need for accurate determination of a potential cancer drug 's dna binding mode and subsequent cellular effects . moreover , dna - destabilizing compounds may be associated with different drug - induced resistance processes . further knowledge of three - dimensional structure activity relationships and the cellular consequences ( cytotoxicity , dna repair processes ) of treatment with dna - destabilizing agents will help to clarify the relevance of cancer drug candidates which stabilize or destabilize the dna helix and will aid the design of potent antitumour agents .
dna targeting drugs represent a large proportion of the actual anticancer drug pharmacopeia , both in terms of drug brands and prescription volumes . 
 small dna - interacting molecules share the ability of certain proteins to change the dna helix 's overall organization and geometrical orientation via tilt , roll , twist , slip , and flip effects . in this ocean of dna - interacting compounds , 
 most stabilize both dna strands and very few display helix - destabilizing properties . 
 these types of dna - destabilizing effect are observed with certain mono- or bis - intercalators and dna alkylating agents ( some of which have been or are being developed as cancer drugs ) . 
 the formation of locally destabilized dna portions could interfere with protein / dna recognition and potentially affect several crucial cellular processes , such as dna repair , replication , and transcription . 
 the present paper describes the molecular basis of dna destabilization , the cellular impact on protein recognition , and dna repair processes and the latter 's relationships with antitumour efficacy .