Analysis of TP53 mutation spectra reveals the fingerprint of the potent environmental carcinogen, aristolochic acid.

Genetic alterations in cancer tissues may reflect the mutational fingerprint of environmental carcinogens. Here we review the pieces of evidence that support the role of aristolochic acid (AA) in inducing a mutational fingerprint in the tumor suppressor gene TP53 in urothelial carcinomas of the upper urinary tract (UUT). Exposure to AA, a nitrophenathrene carboxylic acid present in certain herbal remedies and in flour prepared from wheat grain contaminated with seeds of Aristolochia clematitis, has been linked to chronic nephropathy and UUT. TP53 mutations in UUT of individuals exposed to AA reveal a unique pattern of mutations characterized by A to T transversions on the non-transcribed strand, which cluster at hotspots rarely mutated in other cancers. This unusual pattern, originally discovered in UUTs from two different populations, one in Taiwan, and one in the Balkans, has been reproduced experimentally by treating mouse cells that harbor human TP53 sequences with AA. The convergence of molecular epidemiological and experimental data establishes a clear causal association between exposure to the human carcinogen AA and UUT. Despite bans on the sale of herbs containing AA, their use continues, raising global public health concern and an urgent need to identify populations at risk.

Translesional synthesis past acetylaminofluorene-derived DNA adducts catalyzed by human DNA polymerase kappa and Escherichia coli DNA polymerase IV.

Human DNA polymerase kappa (pol kappa) has a sequence significantly homologous with that of Escherichia coli DNA polymerase IV (pol IV). We used a truncated form of human pol kappa (pol kappaDeltaC) and full-length pol IV to explore the miscoding properties of these enzymes. Oligodeoxynucleotides, modified site-specifically with N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-AF), were used as DNA templates in primer extension reactions that included all four dNTPs. Reactions catalyzed by pol kappaDeltaC were partially blocked one base prior to dG-AAF or dG-AF, and also opposite both lesions. At higher enzyme concentrations, a significant fraction of primer was extended. Analysis of the fully extended reaction product revealed incorporation of dTMP opposite dG-AAF, accompanied by much smaller amounts of dCMP, dAMP, and dGMP and some one- and two-base deletions. The product terminating 3' to the adduct site contained AMP misincorporated opposite dC. On templates containing dG-AF, dAMP, dTMP, and dCMP were incorporated opposite the lesion in approximately equal amounts, together with some one-base and two-base deletions. Steady-state kinetics analysis confirmed the results obtained from primer extension reactions catalyzed by pol kappa. In contract, primer extension reactions catalyzed by pol IV were blocked effectively by dG-AAF and dG-AF. At high concentrations of pol IV, full-length products were formed containing primarily one- or two-base deletions with dCMP, the correct base, incorporated opposite dG-AF. The miscoding properties of pol kappa observed in this study are consistent with mutational spectra observed when plasmid vectors containing dG-AAF or dG-AF are introduced into simian kidney cells [Shibutani, S., et al. (2001) Biochemistry 40, 3717-3722], supporting a model in which pol kappa plays a role in translesion synthesis past acetylaminofluorene-derived lesions in mammalian cells.

Influence of flanking sequence context on the mutagenicity of acetylaminofluorene-derived DNA adducts in mammalian cells.

Site-specifically modified oligodeoxynucleotides were used to explore the influence of neighboring base sequence context on the mutagenic potential of N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-AF) in mammalian cells. Oligodeoxynucleotides ((5)(')TCCTCCTNXNCTCTC, where X is dG-AAF, dG-AF, or dG and N is C, A, G, or T) with different bases flanking the lesion were incorporated into a single-strand shuttle plasmid vector and used to establish the mutational frequency and specificity of dG-AAF and dG-AF adducts in simian kidney (COS-7) cells. Vectors containing dG-AAF promote preferential incorporation of dCMP at the site of the lesion; misincorporation of dAMP and dTMP also was observed. Mutational frequencies range from 11 to 23%. High mutational frequencies (18-23%) were observed when G or T was positioned 5' to dG-AAF and a lower frequency (11%) when C was 5' to the lesion. dCMP was predominantly incorporated opposite the dG-AF adduct when C, A, or T was 5' to the lesion; dAMP and dTMP were misincorporated at a frequency of 2-4%. With G 5' to the lesion, the overall mutational frequency for dG-AF ranged between 11 and 70%; the highest value occurred when C was the 3' flanking base, and the predominant mutation event was G --> T transversion (59%). We conclude from these experiments that dG-AAF and dG-AF promote G --> T transversions and G --> A transitions in mammalian cells. The mutational frequency and specificity of dG-AF vary significantly, depending on the nature of the bases flanking the lesion.

Role for lysine 142 in the excision of adenine from A:G mispairs by MutY DNA glycosylase of Escherichia coli.

MutY participates in the repair of oxidatively damaged DNA by excising adenine from dA:dG and dA:8-oxodG mispairs; this DNA glycosylase can be cross-linked to DNA through Lys-142. We have investigated the properties of a mutant protein in which Lys-142 is replaced by glutamine. Using the rifampicin resistance assay, MutY K142Q was shown to complement the mutY mutator phenotype to the same extent as wild-type MutY. Although MutY K142Q does not form a Schiff base with DNA, it retains in part the catalytic properties of wild-type enzyme. The K142Q mutation selectively impairs processing of DNA containing dA:dG mispairs but not that of substrates containing dA:8-oxodG. Decreased substrate processing is mediated primarily via an increase in K(D) (21.8 nM for MutY vs 298 nM for MutY K142Q). The catalytic constant, measured in single turnover experiments, was not significantly affected. At pH < 6.0, the activity of MutY K142Q on the dA:dG mispair was approximately the same as for wild-type protein, suggesting that a dG(anti) to dG(syn) transition is effected at low pH. The three-dimensional structure of the catalytic domain of MutY K142Q, determined at 1.35 A resolution, shows no significant differences between wild-type and mutant protein, indicating that Lys-142 is not critical for maintaining the conformation of MutY. We conclude that Lys-142 recognizes guanine in the dA:dG mispair, helping position this residue in the syn conformation and facilitating binding of substrate DNA. Lys-142 is not involved in the catalytic steps of base excision.

Escherichia coli responses to a single DNA adduct.

To study the mechanisms by which Escherichia coli modulates the genotoxic effects of DNA damage, a novel system has been developed which permits quantitative measurements of various E. coli pathways involved in mutagenesis and DNA repair. Events measured include fidelity and efficiency of translesion DNA synthesis, excision repair, and recombination repair. Our strategy involves heteroduplex plasmid DNA bearing a single site-specific DNA adduct and several mismatched regions. The plasmid replicates in a mismatch repair-deficient host with the mismatches serving as strand-specific markers. Analysis of progeny plasmid DNA for linkage of the strand-specific markers identifies the pathway from which the plasmid is derived. Using this approach, a single 1, N(6)-ethenodeoxyadenosine adduct was shown to be repaired inefficiently by excision repair, to inhibit DNA synthesis by approximately 80 to 90%, and to direct the incorporation of correct dTMP opposite this adduct. This approach is especially useful in analyzing the damage avoidance-tolerance mechanisms. Our results also show that (i) progeny derived from the damage avoidance-tolerance pathway(s) accounts for more than 15% of all progeny; (ii) this pathway(s) requires functional recA, recF, recO, and recR genes, suggesting the mechanism to be daughter strand gap repair; (iii) the ruvABC genes or the recG gene is also required; and (iv) the RecG pathway appears to be more active than the RuvABC pathway. Based on these results, the mechanism of the damage avoidance-tolerance pathway is discussed.

Mutagenesis induced by a single 1,N6-ethenodeoxyadenosine adduct in human cells.

To study the genotoxic properties of 1,N6-ethenodeoxyadenosine (epsilondA) in human cells, a novel site-specific mutagenesis approach was developed, in which a single DNA adduct was uniquely placed in either strand of a shuttle plasmid vector. The analysis of progeny plasmid derived from the modified strand shows that epsilondA, when incorporated into the position of the second A of 5'-CAA (codon 61 of the ras gene), is mutagenic in human cells, inducing A-->T, A-->G, and A-->C mutations. The efficient induction of A-->T transversions in experiments using modified double- and singlestranded DNA substrates supports the hypothesis that A:T-->T:A transversions in human and animal tumors induced by vinyl compounds reflect misinsertion of dAMP opposite this adduct. Mutagenic events were similar when the adduct was incorporated into either the leading or the lagging strand. EpsilondA was more mutagenic than 8-oxodeoxyguanosine, which induced targeted G-->T transversions in HeLa cells. In Escherichia coli, epsilondA did not significantly miscode (<0.27%) even in the presence of induced SOS functions.

Identification of tamoxifen-DNA adducts in the endometrium of women treated with tamoxifen.

The risk of developing endometrial cancer increases significantly for women treated with tamoxifen (TAM); the present study was designed to investigate the mechanism of this carcinogenic effect. Endometrial tissue was obtained from 16 women treated for varying lengths of time with TAM and from 15 untreated control subjects. DNA was analyzed with a (32)P-post-labeling/HPLC on-line monitoring assay capable of detecting 2.5 adducts/10(10) nucleotides. Using this sensitive and specific assay, TAM-DNA adducts were detected in eight women. The major adducts found were trans and cis epimers of alpha-(N(2)-deoxyguanosinyl) tamoxifen (dG-N(2)-TAM); levels ranged between 0.2-12 and 1.6-8.3 adducts/10(8) nucleotides, respectively. There was marked inter-individual variation in the relative amounts of cis and trans adducts present. Low levels (0.74-1.1 adducts/10(8) nucleotides) of trans and cis forms of dG-N(2)-TAM N-oxide were detected in one patient. DNA adducts derived from 4-hydroxytamoxifen quinone methide were not observed. We conclude from this analysis that trans and cis dG-N(2)-TAMs accumulate in significant amounts in the endometrium of many, but not all, women treated with this drug. The level of adducts found, coupled with the previous demonstration of their mutagenicity [Cancer Res., 59, 2091, 1999], suggest that a genotoxic mechanism may be responsible for TAM-induced endometrial cancer.

Substrate specificity and reaction mechanism of murine 8-oxoguanine-DNA glycosylase.

Genomic DNA is prone to oxidation by reactive oxygen species. A major product of DNA oxidation is the miscoding base 8-oxoguanine (8-oxoG). The mutagenic effects of 8-oxoG in mammalian cells are prevented by a DNA repair system consisting of 8-oxoguanine-DNA glycosylase (Ogg1), adenine-DNA glycosylase, and 8-oxo-dGTPase. We have cloned, overexpressed, and characterized mOgg1, the product of the murine ogg1 gene. mOgg1 is a DNA glycosylase/AP lyase belonging to the endonuclease III family of DNA repair enzymes. The AP lyase activity of mOgg1 is significantly lower than its glycosylase activity. mOgg1 releases 8-oxoG from DNA when paired with C, T, or G, but efficient DNA strand nicking is observed only with 8-oxoG:C. Binding of mOgg1 to oligonucleotides containing 8-oxoG:C is strong (K(D) = 51.5 nm), unlike other mispairs. The average residence time for mOgg1 bound to substrate containing 8-oxoG:C is 18.3 min; the time course for accumulation of the NaBH(4)-sensitive intermediate suggests a two-step reaction mechanism. Various analogs of 8-oxoG were tested as substrates for mOgg1. An electron-withdrawing or hydrogen bond acceptor moiety at C8 is required for efficient binding of mOgg1. A substituent at C6 and a keto group at C8 are required for cleavage. The proposed mechanism of 8-oxoG excision involves protonation of O(8) or the deoxyribose oxygen moiety.

Characterization of a cross-linked DNA-endonuclease VIII repair complex by electrospray ionization mass spectrometry.

Electrospray mass spectrometry techniques were used to characterize components of the active site in Endonuclease VIII by identifying the amino acid sequence and the binding site for a tryptic peptide derived from Endo VIII in a cross-linked DNA-peptide complex. Endo VIII, a DNA repair enzyme with both glycosylase and lyase activities, was covalently bound to a thymidine glycol-containing oligodeoxynucleotide duplex by converting a transient Schiff base formed during the course of the glycosylase activity to a stable covalent bond by chemical reduction with sodium borohydride. After tryptic digestion of the initial product, the identification of the cross-linked peptide was deduced initially from the molecular mass of the tryptic product obtained by negative ion electrospray mass analysis. Nanospray tandem mass spectrometry (MS/MS) analysis of the tryptic product corroborated the molecular mass of the peptide fragment and verified the point of attachment to the oligomer, but failed to produce sufficient fragmentation to sequence the peptide completely. Direct evidence for the amino acid sequence of the peptide was obtained after enzymatic digestion of the DNA portion of the cross-linked DNA-peptide product and analysis by negative ion nanospray MS/MS. Examination of the ions from collision induced fragmentation disclosed that this substance was the N-terminal tryptic fragment of Endo VIII cross-linked to a portion of the oligomer, and that the N-terminal proline from Endo VIII was covalently bound to the residual deoxyribose moiety at the original location of the thymine glycol in the oligomer.

8-oxodGTP incorporation by DNA polymerase beta is modified by active-site residue Asn279.

To understand how the active site of a DNA polymerase might modulate the coding of 8-oxo-7,8-dihydrodeoxyguanine (8-oxodG), we performed steady-state kinetic analyses using wild-type DNA polymerase beta (pol beta) and two active-site mutants. We compared the coding of these polymerases by calculating the ratio of efficiencies for incorporation of dATP and dCTP opposite 8-oxodG and for incorporation of 8-oxodGTP opposite dA and dC. For wild-type pol beta, there is a 2:1 preference for incorporation of dCTP over dATP opposite 8-oxodG using a 5'-phosphorylated 4-base gap substrate. Mutation of either Asn279 or Arg283 to alanine has almost no effect on the ratio. 8-OxodGTP is preferentially incorporated opposite a template dA (24:1) by wild-type pol beta; mutation of Asn279 to alanine results dramatic change whereby there is preferential incorporation of 8-oxodGTP opposite dC (14:1). This suggests that interactions of 8-oxodGTP with Asn279 in the polymerase active site may alter the conformation of 8-oxodGTP and therefore alter its misincorporation.

Comparison of the mutagenic properties of 8-oxo-7,8-dihydro-2'-deoxyadenosine and 8-oxo-7,8-dihydro-2'-deoxyguanosine DNA lesions in mammalian cells.

The comparative mutagenicity of 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) was explored using simian kidney (COS-7) cells. Oligodeoxynucleotides ¿5'-TCCTCCT- G(1)X(2)CCTCTC or 5'-TCCTCCTX(1)G(2)CCTCTC (X = dA, dG, 8-oxodA or 8-oxodG) containing 8-oxodA or 8-oxodG positioned within codon 60 or 61 of the non-coding strand of human c-Ha-ras1 gene were inserted into a single-stranded phagemid shuttle vector. The vector was replicated in COS-7 cells and the progeny plasmids were used to transform Escherichia coli DH10B. The transformants were analyzed by oligodeoxynucleotide hybridization and DNA sequence analysis to establish the mutation frequency and specificity. When 8-oxodA was positioned at X(1), targeted A(oxo)-->C transversions were detected; the mutation frequency was 1.2%. When 8-oxodA was positioned at X(2), one targeted mutant among 416 colonies screened (an A(oxo)-->G transition) was detected. Thus, the mutation frequency and spectrum of 8-oxodA depend on the sequence context of the lesion. The mutation frequency of 8-oxodG at X(1) and X(2) was 5.2 and 6.8%, respectively. G(oxo)-->T transversions dominated the spectrum, accompanied by small numbers of G(oxo)-->A transitions and G(oxo)-->C transversions. We conclude that 8-oxodA has mutagenic potential in mammalian cells, generating A-->C transversions. However, when tested under similar conditions, the mutation frequency of 8-oxodA is at least four times lower than that of 8-oxodG.

3,N(4)-ethano-2'-deoxycytidine: chemistry of incorporation into oligomeric DNA and reassessment of miscoding potential.

3,N(4)-Ethano-2'-deoxycytidine (ethano-dC) may be incorporated successfully into synthetic oligodeoxynucleotides by omitting the capping procedure used in the automated DNA synthetic protocols immediately after inserting the lesion and in all iterations thereafter. Ethano-dC is sensitive to acetic anhydride found in the capping reagent, and multiple oligomeric products are formed. These products were identified by examining the reaction of ethano-dC with the capping reagent, and several acetylated, ring-opened products were characterized by electrospray mass spectrometry and collision induced dissociation experiments on a tandem quadrupole mass spectrometer. A scheme for the formation of the acetylated products is proposed. In addition, the mutagenic profile of ethano-dC was re-examined and compared to that for etheno-dC. Ethano-dC is principally a blocking lesion; however, when encountered by the exo(-)Klenow fragment of DNA polymerase, dAMP (22%), TMP (16%), dGMP (5.3%) and dCMP (1.2%) were all incorporated opposite ethano-dC, along with an oligomer containing a one-base deletion (0.6%).

Mutagenesis of the N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine DNA adduct in mammalian cells. Sequence context effects.

Site-specifically modified oligodeoxynucleotides were used to investigate the mutagenic properties of a major cooked food mutagen-derived DNA adduct, N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (dG-C8-PhIP). dG-C8-PhIP-modified oligodeoxynucleotides were prepared by reacting an oligodeoxynucleotide containing a single dG (5'-TCCTCCTXGCCTCTC, where X = C, A, G, or T) with N-acetoxy-PhIP. The unmodified and dG-C8-PhIP-modified oligomers were inserted into single-stranded phagemid vectors. These single-stranded vectors were transfected into simian kidney (COS-7) cells. The progeny plasmid obtained was used to transform Escherichia coli DH10B. When dC was at the 5'-flanking position to dG-C8-PhIP, preferential incorporation of dCMP, the correct base, was observed opposite the dG-C8-PhIP. Targeted G --> T transversions were detected, along with lesser amounts of G --> A transitions and G --> C transversions. No mutations were detected for the unmodified vector. The influence of sequence context on the dG-C8-PhIP mutation frequency and spectrum was also explored. When the dC 5'-flanking base was replaced by dT, dA, or dG, the mutational spectra were similar to that observed with dC-flanking base. Higher mutational frequencies (28-30%) were observed when dC or dG was 5' to dG-C8-PhIP. A lower mutational frequency (13%) was observed when dA was at the 5' to the lesion. Single-base deletions were detected only when dG or dT flanked the adduct. We conclude that dG-C8-PhIP is mutagenic, generating primarily G --> T transversions in mammalian cells. The mutational frequency and specificity of dG-C8-PhIP vary depending on the neighboring sequence context.

Tamoxifen-DNA adducts detected in the endometrium of women treated with tamoxifen.

Women treated for breast cancer with tamoxifen are at increased risk of developing endometrial cancer. This carcinogenic effect has been attributed to estrogenic stimulation and/or to a genotoxic effect of this drug. To examine genotoxicity, we developed a (32)P-postlabeling TLCL/HPLC procedure for quantitative analysis of tamoxifen-DNA adducts in endometrial tissue. This assay is several orders of magnitude more sensitive than those previously used for this purpose; with it, we can detect five tamoxifen-DNA adducts in 10(11) bases. Endometrial tissue was obtained from women undergoing tamoxifen therapy and from untreated control subjects. DNA adducts, identified as trans and cis epimers of alpha-(N(2)-deoxyguanosinyl)tamoxifen, were detected in six of thirteen patients in the tamoxifen-treated group. Levels of trans and cis adducts ranged from 0.5 to 8.3 and from 0.4 to 4.8 adducts/10(8) nucleotides, respectively. Tamoxifen-DNA adducts were not detected in endometrial tissue obtained from the control subjects. We conclude from this study that one or more tamoxifen metabolites react with endometrial DNA to form covalent adducts, establishing the potential genotoxicity of this drug for women and suggesting the use of TAM-DNA adducts as biomarkers for investigations of tamoxifen-induced endometrial cancer.

Mutagenic properties of the 8-amino-2'-deoxyguanosine DNA adduct in mammalian cells.

The DNA adduct 8-amino-2'-deoxyguanosine (8-amino-dG) is found in liver DNA of rats treated with the hepatocarcinogen 2-nitropropane. Site-specifically modified oligodeoxynucleotides were used to explore the mutagenic potential of 8-amino-dG in simian kidney (COS-7) cells. Oligodeoxynucleotides (5'-TCCTCCTX1G2CCTCTC and 5'-TCCTCCTG1X2CCTCTC, X = dG or 8-amino-dG) with the lesion positioned at codon 60 or 61 of the non-coding strand of the human c-Ha- ras1 gene were inserted into single-stranded phagemid vectors and transfected into COS-7 cells. The progeny plasmid obtained was used to transform Escherichia coli DH10B. Transformants were analyzed by oligodeoxynucleotide hybridization and DNA sequencing to establish the mutation frequency and spectrum produced by the modified base. The correct base, dCMP, was incorporated preferentially opposite 8-amino-dG at X1and X2. When 8-amino-dG was at X1, targeted GNH2-->T transversions were detected, along with smaller numbers of GNH2-->A transitions and GNH2-->C transversions. When the adduct was at X2, only GNH2-->T transversions were observed. The frequencies of targeted mutation at X1and X2were 2.7 and 1.7%, respectively. Mutation frequency and mutagenic spectrum were sequence context dependent. In addition, non-targeted G-->T transversions, accompanied by some G-->A transitions, were detected 5' to 8-amino-dG when the lesion was at X2. We conclude that 8-amino-dG is a mutagenic lesion, generating G-->T and G-->C transversions and G-->A transitions in mammalian cells.

Cellular response to exocyclic DNA adducts.

The mutagenic potential of three exocyclic DNA adducts was studied in Escherichia coli and simian kidney cells by incorporating them into single-stranded DNA. Differences in the mutagenic potency of the adducts were observed between hosts: 1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine were more mutagenic in simian cells, whereas 1,N2-(1,3-propan-1,3-diyl)-2'-deoxyguanosine was more mutagenic in E. coli. To investigate the cellular response to DNA adducts, a double-stranded DNA vector system was developed. Use of this system showed that 1,N6-ethenodeoxyadenosine blocks DNA synthesis strongly, and DNA synthesis past this adduct was highly accurate in E. coli. The blockage of DNA synthesis was overcome in an error-free manner by the recombination repair mechanism (daughter-strand gap repair).

Mutagenic specificity of (acetylamino)fluorene-derived DNA adducts in mammalian cells.

Site-specifically modified oligodeoxynucleotides were used to explore the mutagenic potential of dG-AAF and dG-AF adducts in mammalian cells. The miscoding properties of these arylamine adducts were established by analyzing fully extended products of primer extension reactions catalyzed by mammalian DNA polymerases alpha, beta, and delta. On DNA templates containing dG-AAF, pol alpha generated two-base deletions and promoted incorporation of small amounts of dCMP, dAMP, and dTMP opposite the lesion. Reactions with pol beta were associated exclusively with two-base deletions. Primer extension catalyzed by pol delta was strongly blocked by the adduct. On DNA templates containing dG-AF, all three DNA polymerases generated full-length products, preferentially incorporating dCMP opposite the lesion. A single-stranded shuttle vector containing 5'TCCTCCTCXCCTCTC (X = dG-AAF, dG-AF, or dG) was used to establish the frequency and specificity of dG-AAF- and dG-AF-induced mutations in simian kidney (COS-7) cells. Vectors containing a single dG-AAF or dG-AF adduct promote significant incorporation of dAMP and lesser amounts of dTMP opposite the lesion. dG-AAF also promoted some incorporation of dGMP and a two-base deletion. dG-AAF was 3.8 times more mutagenic than dG-AF (11% vs 2.9%) in COS cells. We conclude from this study that dG-AAF and dG-AF produce G --> T transversions and, to a much lesser degree, G --> A transitions in mammalian cells.

MutY DNA glycosylase: base release and intermediate complex formation.

MutY protein, a DNA glycosylase found in Escherichia coli, recognizes dA:dG, dA:8-oxodG, and dA:dC mismatches in duplex DNA, excising the adenine moiety. We have investigated the mechanism of action of MutY, addressing several points of disagreement raised by previous studies of this enzyme. MutY forms a covalent intermediate with its DNA substrate but does not catalyze strand cleavage. The covalent intermediate has a half-life of approximately 2.6 h, 2 orders of magnitude greater than the half-life of Schiff bases formed when E. coli formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III react with their respective substrates. The covalent complex between MutY and its DNA substrate involves Lys-142; however, the position of this residue in the presumptive active site differs from that of catalytic residues involved in Schiff base formation associated with endonuclease III and related DNA glycosylases/AP lyases. MutY converts DNA duplexes containing the dA:8-oxodG mispair to a product containing an abasic site; heat-induced cleavage of this product may account for the several reports in the literature that ascribe AP lyase activity to MutY. The MutY-DNA intermediate complex is highly stable and hinders access by Fpg to DNA, thereby avoiding a double-strand break. Cross-linking of MutY to DNA may play an important role in the regulation of base excision repair.