the recognition of specific dna sequences or structures by dna - binding proteins is a crucial regulatory step during dna replication , control of gene expression , and the response to and repair of dna damage . protein and dna damage induced by endogenous electrophiles is associated with a number of human diseases , including cancer , cardiovascular disease , and neurological disorders ( 13 ) . for example , oxidation products of polyunsaturated fatty acids comprise a series of cytotoxic ,-unsaturated aldehydes , including malondialdehyde , 4-hydroxynonenal , and acrolein , that readily form covalent adducts with cellular macromolecules , contributing to their toxicity ( reviewed in ref ( 4 ) ) . acrolein is a mutagen and a tumor initiator produced endogenously via lipid peroxidation and myeloperoxidase - catalyzed amino acid oxidation ( 58 ) . it is also an ubiquitous pollutant found in automobile exhaust and cigarette smoke ( 9 ) . acrolein exhibits facile reactivity with proteins ( 8) and represents a significant source of endogenous dna damage by forming cyclic adducts with dna bases and dnaprotein cross - links ( 10,11 ) . the major products of the reaction of acrolein with dna are exocyclic adducts of deoxyguanosine , ( 8r / s)-3-(2-deoxyribos-1-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2a]purin-10(3h)-one ( -hopdg)1 and ( 6r / s)-3-(2-deoxyribos-1-yl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2a]purin-10(3h)-one ( -hopdg ) ( 10 ) ( figure 1 ) . -hopdg is more mutagenic than -hopdg and induces gt transversions in site - specific mutagenesis experiments ( 12 ) . both -hopdg and -hopdg are present in human lung dna at levels in excess of those typically reported for polycyclic aromatic hydrocarbondna adducts ( 13 ) . 1abbreviations : bsa , bovine serum albumin ; edta , ethylenediaminetetraacetic acid ; -hopdg , -hydroxy-2-propanodeoxyguanosine ; m1dg , pyrimido[1,2-]purin-10(3h)-one ; mops , 4-morpholinepropanesulfonic acid ; ms , mass spectrometry ; ner , nucleotide excision repair ; sds - page , sodium dodecyl sulfatepolyacrylamide gel electrophoresis . abbreviations : bsa , bovine serum albumin ; edta , ethylenediaminetetraacetic acid ; -hopdg , -hydroxy-2-propanodeoxyguanosine ; m1dg , pyrimido[1,2-]purin-10(3h)-one ; mops , 4-morpholinepropanesulfonic acid ; ms , mass spectrometry ; ner , nucleotide excision repair ; sds - page , sodium dodecyl sulfatepolyacrylamide gel electrophoresis . structures of dna adducts and their ring - opening products . while -hopdg contributes to acrolein toxicity by directly affecting dna replication ( 12,14 ) , the adduct itself is a reactive molecule . in duplex dna , -hopdg undergoes a spontaneous ring - opening reaction to generate n - oxopropyl-2-deoxyguanosine that can react with primary amines on cellular macromolecules ( 15 ) . -hopdg positioned within a cp*g sequence context in duplex dna undergoes ring opening and subsequent reaction with a guanine base in the complementary strand to form interstrand dnadna cross - links ( 16 ) . the ring - opened -hopdg adduct is also reactive toward amino groups of peptides and is capable of forming reversible schiff base - mediated cross - links to proteins ( 12 ) . a role for dnaprotein cross - links during acrolein toxicity has not been fully elucidated . the interaction of adducted oligonucleotides with restriction endonucleases represents a useful model system with which to evaluate the impact of dna damage on recognition and catalysis of dna - binding proteins . previously , our group showed that exocyclic adducts in an ecori recognition site blocked binding and cleavage of dna by that enzyme , although ring - opened adducts were substrates for endonucleolytic cleavage by ecori ( 17 ) . here , we examine the fate of -hopdg and consequently its ring - opened analogue , when incorporated into duplex substrates of ecori . we describe the ability of -hopdg to cross - link to ecori and affect enzyme activity . the results suggest that the dnaprotein cross - link is unstable and either undergoes hydrolysis to free oligonucleotide and enzyme or can be reduced to a stable , irreversible cross - link . t4 polynucleotide kinase and pvuii were obtained from new england biolabs ( beverly , ma ) . ecori for some cross - linking experiments was purchased from mbi fermenetas ( hanover , md ) . the 21-mer oligonucleotides contained a unique ecori restriction site ( 5-gaattc-3 ) in the sequence 5-tatcatgtctnaattcccggt-3 , where n = dg , -hopdg , or pyrimido[1,2-]purin-10(3h)-one ( m1dg ) . the sequence of the complementary 27-mer oligonucleotide was 5-agaccgggaattcagacatgatacgga-3. the scrambled ecori sequence was 5-tatcatgtctntatacccggt-3 , where n = -hopdg ; it was annealed to its complementary sequence . the m1dg - modified oligonucleotide was synthesized using the method previously described ( 20 ) and purified by polyacrylamide gel electrophoresis ( 21 ) . unmodified 21-mer and 27-mer oligonucleotides were synthesized by integrated dna technologies ( coralville , ia ) . modified and unmodified 21-mer oligonucleotides were 5-end - labeled in a phosphorylation buffer of 50 mm 4-morpholinepropanesulfonic acid ( mops ) ( ph 7.3 ) , 10 mm mgcl2 , 100 m edta , and 5 mm dithiothreitol containing 5 m oligonucleotide , 0.5 mm [ p]-atp , and 10 units of t4 polynucleotide kinase . oligonucleotides were purified from free [ p]-atp by passage through bio - spin p-6 columns ( bio - rad , hercules , ca ) . for restriction endonuclease studies , substrates were generated by annealing radiolabeled -hopdg- or m1dg - modified 21-mer ( 2 pmol ) to a complementary 27-mer ( 10 pmol ) in 50 mm mops ( ph 7.4 ) and 100 mm nacl ( 50 l total volume ) by heating to 90 c for 3 min , followed by slow cooling to room temperature . dnaprotein cross - links were detected essentially as described with slight modifications ( 12 ) . p - labeled substrate ( 160 pm ) was incubated with ecori or pvuii restriction enzyme ( 1.6 nm ) or bovine serum albumin ( bsa ) ( 5000 nm ) in 50 mm mops ( ph 7.4 ) , 10 mm nacl , and 0.1 mm ethylenediaminetetraacetic acid ( edta ) , at room temperature for 2 h. nacnbh3 ( 50 mm ) was added immediately following the addition of enzyme . because mg is required for ecori cleavage activity but not required for the specific binding of enzyme to dna , mg was omitted from the incubation . the reactions were terminated by addition of laemmli sample buffer , then boiled for 5 min , and separated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis ( sds - page ) ( 22 ) . radiolabeled substrates were generated by annealing adducted or unmodified 5-p - labeled 21-mer oligonucleotide with an equimolar amount of 5-p - labeled 27-mer complement . annealing of 2.5 pmol of each oligonucleotide was performed in buffer ( 50 mm mops , ph 7.4 , and 100 mm nacl ) by heating to 90 c for 3 min , followed by slow cooling . nonradiolabeled substrates were generated by annealing adducted or unmodified 21-mer with complementary oligonucleotide under the conditions described above . restriction digests ( 10 l ) contained 5 nm p - labeled substrate , 495 nm unlabeled substrate , 50 mm mops ( ph 7.9 ) , 10 mm mgcl2 , 100 mm nacl , and 5 nm ecori restriction endonuclease . the reactions were carried out for specified times at 37 c , then quenched by the addition of 10 mm edta in 90% formamide . the products of the reaction were resolved on a 20% denaturing gel using the ultrapure sequagel system ( national diagnostics , atlanta , ga ) . the 21-mer oligonucleotide 5-cleavage product was an 11-mer , whereas the 27-mer substrate s 5-cleavage product was an 8-mer . thus , by monitoring the appearance of the 11-mer and the 8-mer , cleavage of both strands of the substrate duplex could be monitored simultaneously . the extent of cleavage was determined by integrating the radioactivity of each band , then dividing the radioactivity of the band corresponding to cleavage product by the sum of the radioactivity of cleaved and uncleaved oligonucleotide to give the percent cleavage . in some experiments , incubation mixtures were prepared in which 500 nm unlabeled adducted substrate or unmodified substrate was incubated for 2 h with 5 nm ecori . then , 500 nm unmodified substrate , spiked with p - labeled unmodified substrate , was added to the reaction mixture , and cleavage was monitored for 2 h. for other experiments , reactions were prepared under noncleavage conditions , that is , in the absence of mg . p - labeled dna and 495 nm nonradiolabeled dna ) was incubated in mg - free buffer ( 50 mm mops , ph 7.9 , and 100 mm nacl ) for 2 h with ecori ( 5 nm ) in the presence or absence of nacnbh3 . following preincubation , 10 mm mgcl2 was added to the reaction mixture , and cleavage was monitored for 1 h. ecori binds the palindromic sequence , 5-gaattc-3 , and sequentially cleaves both strands of the dna duplex at the phosphodiester bond positioned 3 to deoxyguanosine . p - labeled oligonucleotide duplexes were prepared containing a unique ecori recognition site that was modified with -hopdg . substrate dna was incubated for 2 h with ecori ( molar ratio of dna duplex : ecori = 1:10 ) in the presence of nacnbh3 , as described in the . nacnbh3 does not react with ecori or -hopdg but is capable of reducing an imine cross - link between the free aldehyde form of -hopdg and a lysine residue . ecori reacted with -hopdg - modified substrates to form a dnaprotein cross - link observable by sds - page analysis ( figure 2 ) . formation of the gel - shifted band corresponding to the proteindna complex was dependent on stabilization of the cross - link by reduction with nacnbh3 . a weaker , second band was observed that migrated more slowly than the major -hopdgecori band . p - labeled -hopdg- or m1dg - modified or unmodified dna substrates ( 160 pm ) were incubated with ecori ( 1.6 nm ) at room temperature for 2 h in the presence or absence of 50 mm nacnbh3 . -hopdg ring opens to form an aldehydic moiety that is capable of reacting with proteins ( 15 ) . the malondialdehydedeoxyguanosine adduct , m1dg , similarly ring opens in duplex dna to form a potentially reactive oxopropenyl moiety , n - oxopropenyl - dg ( 23 ) . the presence of m1dg in the ecori recognition site does not prevent recognition of the site by ecori ( 17 ) . therefore , we tested whether m1dg is also able to form cross - links to ecori . in contrast to -hopdg , no cross - link was observed when m1dg - modified substrates were incubated with ecori with or without nacnbh3 reduction ( figure 2 ) . dna substrates were prepared in which -hopdg was positioned in a scrambled ecori recognition site ( 5-gtatac-3 ) . no ecoridna cross - link was observed when -hopdg was in the scrambled sequence ( figure 3 ) . when ecori was inactivated by heat ( 65 c for 20 min ) prior to the reaction with -hopdg - modified substrate , only an extremely faint band at the position of the dnaprotein cross - link was detected after 2 h ( figure 4 ) . furthermore , -hopdg did not cross - link to a restriction enzyme , pvuii , that does not specifically recognize the sequence of dna where the adduct was positioned . also , -hopdg did not form cross - links with bsa , a common stabilizing agent added to commercial preparations of ecori , even at a dna : bsa molar ratio = 1:5000 ( data not shown ) . these results indicate that the cross - link formed between -hopdg and ecori is most likely formed as a result of specific binding of the ecori recognition sequence and probably involves amino acids in close proximity to the adduct in the dnaprotein complex . p - labeled -hopdg - modified or unmodified dna substrates ( 160 pm ) were incubated with ecori ( 1.6 nm ) at room temperature for 2 h in the presence or absence of nacnbh3 . -hopdg was positioned in either the ecori restriction site ( gaattc ) or a scrambled sequence ( gtatac ) . ecori ( 1.6 nm ) , native or denatured at 65 c for 20 min , was incubated with -hopdg - modified p - end - labeled dna substrate ( 160 pm ) at room temperature for 2 h. nacnbh3 ( 50 mm ) was added to the reaction to stabilize the schiff base linkage between protein and dna . reactions were prepared in the presence of nacnbh3 , and then , a second reducing agent , nabh4 , was added to rapidly quench any remaining aldehydic substrate at specific time points . the -hopdgecori cross - link was observed within 10 min . the conversion of free dna to cross - linked product reached a plateau within 3 h. time course of -hopdg cross - linking to ecori . -hopdg - modified p - end - labeled dna substrate ( 160 pm ) was incubated with ecori ( 1.6 nm ) for up to 5 h at room temperature in the presence of 50 mm nacnbh3 , and then , a second reducing agent , nabh4 ( 100 mm ) , was added to rapidly quench any remaining aldehydic substrate at specific time points . a representative autoradiogram from three independent experiments is presented , which displays the accumulation of the dnaprotein cross - linked band . oligonucleotide duplexes containing a single adduct located within a unique ecori recognition site were 5-p - end - labeled on both strands . the adducted strand of the duplex was a different length than its complement , and the oligonucleotides were designed to release p - end - labeled cleavage products of different lengths . the complement strand of the -hopdg - modified substrate was cleaved nearly as efficiently as unmodified substrate ( 90% ) ( figure 6b ) . in contrast , only 20% of the -hopdg - modified strand was cleaved by ecori within 2 h. effect of -hopdg on ecori activity . ( b ) cleavage reactions containing 5 nm p - labeled oligonucleotide duplex , 495 nm nonlabeled oligonucleotide substrate , and 5 nm ecori were incubated at 37 c for various times , then quenched , and subjected to gel electrophoresis and phosphorimager analysis as described in the . unmodified substrate , 27-mer strand (  ) ; -hopdg - modified substrate , 27-mer strand (  ) ; -hopdg - modified substrate , 21-mer strand (  ) . ( c ) nonlabeled oligonucleotide duplexes modified with -hopdg or unmodified duplexes ( 500 nm ) were incubated with ecori endonuclease ( 5 nm ) for 2 h at 37 c , prior to the addition of fresh unmodified ecori substrate , spiked with p - labeled unmodified substrate , to a final concentration of 500 nm . cleavage of the radiolabeled substrate was monitored for 2 h. the data shown represent cleavage of the 27-mer strand of the radiolabeled duplex in the presence of the indicated competitor substrate . the 21-mer strand of each duplex was cleaved similarly to its complement ( data not shown ) . unmodified competitor substrate (  ) ; -hopdg - modified competitor substrate (  ) . to examine whether the reduced cleavage efficiency of modified substrate was due to a sequestration or inactivation of ecori by -hopdg , nonradiolabeled -hopdg - adducted substrates or unadducted substrates were incubated with ecori for 2 h. then , p - labeled unmodified substrate was added to the reaction mixture , and cleavage was monitored . the radiolabeled unmodified substrate was cleaved to a similar extent , regardless of whether unmodified substrate or -hopdg - modified substrate was initially incubated with the enzyme ( figure 6c ) . thus , ecori retained complete activity following exposure to the dna adduct . to examine whether a stabilized cross - link could affect ecori activity , -hopdg - modified or unmodified substrates were incubated with ecori in the absence of mg and either the presence or the absence of nacnbh3 for 2 h ; then , mgcl2 was added to the reaction mixture to initiate cleavage . following mgcl2 addition , ecori cleavage of the substrate was substantially decreased in samples containing -hopdg - modified dna and nacnbh3 as compared to samples containing -hopdg - modified substrate in the absence of reducing agent . there was no effect of nacnbh3 on cleavage of the unmodified dna substrate by ecori ( figure 7 ) . p - radiolabeled -hopdg - modified substrate or unmodified substrate ( 500 nm ) was incubated with ecori ( 5 nm ) in the absence of mg and either the presence or the absence of nacnbh3 for 2 h. following preincubation , mgcl2 ( 10 mm ) was added to initiate cleavage . unmodified substrate , nacnbh3 (  ) ; unmodified substrate , + nacnbh3 (  ) ; -hopdg - modified substrate , nacnbh3 (  ) ; and -hopdg - modified substrate , + nacnbh3 incubation (  ) . the identification of cellular targets of endogenous reactive aldehydes has garnered significant attention in an attempt to understand the genotoxic and cytotoxic mechanisms of these molecules . the products of acrolein s reaction with cellular macromolecules have been suggested to participate in diseases associated with oxidative stress ( 24 ) . in addition to forming adducts to protein and adducts to dna , acrolein induces schiff base - mediated cross - link formation between dna and protein ( 11 ) , and the major acroleindna adduct -hopdg is itself reactive with both dna and proteins ( 12,16,2527 ) . thus , -hopdg may represent a mode for acrolein s dnaprotein cross - link formation in vivo . although a mechanistic link between dnaprotein cross - links and disease has not been elucidated , the formation of cross - links can be detrimental to cell survival . the bulky nature of the cross - links is likely to disrupt dna metabolism by posing significant blocks to replication and transcription . consistent with this proposal , aldehyde - derived dnaprotein cross - links block sv40 dna replication ( 28 ) . although little is known about the mutagenic properties of dnapeptide cross - links , minko et al . recently described the mutagenic potential of nacnbh3-reduced-hopdg - mediated cross - links to peptides ( 29 ) . in the present study , we demonstrated the ability of -hopdg to cross - link to a protein that forms intimate interactions with dna , the restriction endonuclease , ecori . previous work has demonstrated that -hopdg - containing oligonucleotides form cross - links to histone and to t4 pyrimidine dimer dna glycosylase ( 12 ) . it appears that -hopdg forms cross - links to ecori more rapidly and at a considerably reduced excess of protein to dna . the differences between the two studies are most likely attributable to the fact that ecori has high affinity and specificity for binding duplex oligonucleotides that contain a recognition site and that the -hopdg adduct is present at the first dg residue in the recognition site . given that ecori was able to recognize and digest -hopdg - modified dna substrates , -hopdg is presumed to be accommodated in the recognition interface in a manner similar to physiological substrates . furthermore , the native structure of ecori was essential for rapid dnaprotein cross - link formation , and no cross - link was detected when -hopdg was not located within the enzyme s recognition site . these results suggest that upon binding to the -hopdg - modified cognate site , the reactive n - oxopropyl moiety forms a cross - link between the 2-amino position of the modified g ( naattc ) and a proximal amino acid at the recognition interface . lysine residues have been implicated in dnaprotein cross - link formation by acetaldehyde ( 30 ) and may also participate in -hopdg - mediated cross - links . the stabilization of the ecoridna cross - links by nacnbh3 is consistent with this in that nacnbh3 preferentially reduces imine bonds formed by reaction of carbonyl groups with lysine residues ( 31 ) . there are several amino acids involved in the interaction of ecori with its recognition sequence , which contain primary amines that could be involved in cross - link formation . although lys-148 , lys-117 , and lys-89 are in the vicinity , a likely candidate is lys-113 , which not only polarizes one of the oxygens of the scissile phosphate but also , along with arg-145 , may stabilize the phosphorane intermediate in the transition state ( 32 ) . the lys-113 side chain is disordered in the apoenzyme but becomes ordered upon binding dna substrate ( 33 ) . in molecular dynamics simulations , lys-113 moves to different positions depending on the presence or absence of mg(32 ) . the conditions used for the enzymatic cleavage assay provided dna substrate in 100-fold molar excess to ecori . nevertheless , no loss of activity was observed without nacnbh3 reduction and stabilization of the cross - link ; treatment of ecori alone with nacnbh3 did not reduce endonucleolytic activity . the loss of activity observed following treatment of the ecori-hopdgdna complex with nacnbh3 correlates to the appearance of the stable cross - link band on gel electrophoresis . thus , it appears that the formation of the putative imine - mediated proteindna cross - link is readily reversible so that the proteindna cross - link hydrolyzes to ecori and -hopdg - containing oligonucleotide during the time course of the incubation . the proposed mechanism for -hopdg - mediated formation of dnaprotein cross - links involves ring opening of -hopdg to n-(-oxopropyl)-dg , followed by reaction with a primary amine . kurtz and lloyd observed that -hopdg cross - links occurred only with the nh2 termini of peptides containing lysine and arginine residues ( 27 ) . this is consistent with the lower pka and stronger nucleophilic nature of an -amine as compared to the -amine of lysine . in related chemistry , the aldehydic form of an abasic site cross - linked to the catalytically active n - terminal threonine of t4 pdg and the imine was trapped by nacnbh3 reduction ( 34 ) . it is unlikely that the -hopdgecori cross - link occurs at the nh2 terminus of the protein . a revised model of ecori in solution places the n - terminal regions at a subunit interface distal to the dna , and removal of the first four residues has no effect on catalytic activity , suggesting that the n termini are spatially remote from the ecori active sites ( 35,36 ) . it is more likely that cross - linking occurs to a lys residue in the interior of the protein . exclusion of water from the recognition interface could lower the pka of a lys side chain , thereby increasing the fraction of deprotonated nucleophilic amine . -hopdg readily formed cross - links to ecori , which were trappable by nacnbh3 reduction , but the m1dg adduct did not . although m1dg undergoes rapid hydrolytic ring opening in duplex dna , the aldehyde of the product , n-(3-oxopropenyl)-dg , is conjugated to the exocylic amino group , which reduces its reactivity [ i.e. , n-(3-oxopropenyl)-dg could be considered a vinylogous amide ] ( 37 ) . furthermore , n-(3-oxopropenyl)-dg exhibits a pka of 6.9 so that at physiological ph it exists predominantly as the negatively charged conjugate base ( 37,38 ) . thus , m1dg does not appear to be a good candidate to form cross - links with dna binding proteins . malondialdehyde - mediated dnaprotein cross - links have been documented in vitro and in cells treated with mda ( 39 ) . in that study , cross - linking only occurred if the proteinmda adduct was formed first and then exposed to dna . this behavior is consistent with the present observation that a dna molecule containing the malondialdehyde adduct , m1dg , does not form cross - links to ecori . although -hopdgprotein cross - links can be generated and stabilized in vitro , the reversibility of the cross - link may limit its effects in vivo . in cos-7 cells , replication of plasmid dna adducted with a nacnbh3-reduced--hopdg - peptide cross - link resulted in approximately 8.4% mutation frequency , consisting of primarily g to t transversions , which is a higher frequency of mutation than that observed following replication of the -hopdg adduct ( 29 ) . the excision of dnaprotein cross - links by the uvrabc system was shown to proceed with moderate efficiency ( 40 ) . thus , secondary modifications to the adduct structure are capable of modulating the mutagenicity of -hopdg and underscore the likelihood that dna replication , transcription , or repair may be disrupted dependent on the longevity of the -hopdgprotein cross - link . to date , the stability of schiff base - mediated cross - links in vivo has not been elucidated . it will be interesting to determine what biological role , if any , -hopdg - mediated dnaprotein cross - links play in acrolein - mediated toxicity in vivo .
the toxicity of acrolein , an ,-unsaturated aldehyde produced during lipid peroxidation , is attributable to its high reactivity toward dna and cellular proteins . 
 the major acroleindna adduct , -hydroxypropano-2-deoxyguanosine ( -hopdg ) , ring opens to form a reactive n2-oxopropyl moiety that cross - links to dna and proteins . 
 we demonstrate the ability of -hopdg in a duplex oligonucleotide to cross - link to a protein ( ecori ) that specifically interacts with dna at a unique sequence . the formation of a cross - link to ecori was dependent on the intimate binding of the enzyme to its -hopdg - modified recognition site . 
 interestingly , the cross - link did not restrict the ability of ecori to cleave dna substrates . however , stabilization of the cross - link by reduction of the schiff base linkage resulted in loss of enzyme activity . 
 this work indicates that the -hopdgecori cross - link is in equilibrium with free oligonucleotide and enzyme . 
 reversal of cross - link formation allows ecori to effect enzymatic cleavage of competitor oligonucleotides .