Release of N2,3-ethanoguanine from haloethylnitrosourea-treated DNA by Escherichia coli 3-methyladenine DNA glycosylase II

3-Methyladenine DNA glycosylase II (Gly II), purified from Escherichia coli cells which carry the plasmid PYN1000, has been tested for its ability to release N2,3-ethanoguanine from DNA modified by the antitumor agent N-[2-chloroethyl-1,2-14C]-N'-cyclohexyl-N-nitrosourea ([14C]CCNU). Gly II has been shown to release N2,3-ethanoguanine in a protein- and time-dependent manner at a rate that exceeds the rate at which this enzyme releases other alkylated bases from [14C]CCNU-modified DNA. This finding widens the known substrate specificity for Gly II to include a modified base which bears an exocyclic ring structure, a class of modifications caused by a variety of chemical carcinogens.     

Release of 7-alkylguanines from N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-modified DNA by 3-methyladenine DNA glycosylase II

Purified bacterial 3-methyladenine DNA glycosylase II releases four 7-alkylguanines from [3H]N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-modified DNA: 7-(2-hydroxyethyl)guanine,1,2-bis(7-guanyl)ethane, 7-(2-chloroethyl)guanine, and 7-(2-ethoxyethyl)guanine. 7-(2-Ethoxyethyl)guanine, a new compound, is formed as a result of an interaction with ethanol, a common solvent for the 2-haloethylnitrosoureas. Of the four 7-alkylguanines which are released from [3H]N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-modified DNA, 7-(2-hydroxyethyl)guanine is released at a rate very much slower than the other three. As shown by a study of the spontaneous decomposition of the corresponding 7-alkyl-deoxyguanines, differences in chemical stability do not appear to explain the slow release of 7-(2-hydroxyethyl)guanine. In view of previous results showing a difference in the distribution of alkylation products between sensitive and resistant glial cell lines, the broad specificity of this enzyme suggests that glycosylase activity could play a role in cellular resistance to 2-haloethylnitrosoureas.     

The release of 3-methyladenine from nucleosomal DNA by a 3-methyladenine DNA glycosylase

Nucleosomes from chicken erythrocytes, with DNA containing anaverage of 144 base pairs, were alkylated with [³H]methylnitrosourea.The level of alkylation of the nucleo-somal DNA was 48% of thatof free DNA. The histones had approximately one tenth the radioactivityof the DNA. There was no statistically significant differencebetween alkylation of nucleosome bases in the major vs. minorgroove. When the first 50 residues of the alkylated nudeosomalDNA were examined on sequencing gels, the 7-methylguanine and3-methyladenine (3-MeA) residues were distributed randomly.The 3-MeA DNA glycosylase I of E. coli was used to measure therelease of 3-MeA from nudeosomal DNA. Incubation at 37°Cresulted in a release which reached a plateau of {small tilde}33%of the 3-MeA groups of the nudeosomal DNA. A partially purified3-MeA DNA glycosylase from rat liver gave similar results. Thelimited enzymatic release is most likely due to steric hindranceof the enzyme by the DNA-histone interactions on the surfaceof the core partide. An alteration of nudeosomal conformationhas been suggested as an explanation for repair of nudeosomalDNA. Two model systems have been examined. The addition of ethidiumbromide to alkylated nudeosomes increased the enzymatic releaseof 3-MeA to {small tilde}75% and altered the electron microscopicappearance. The chemical alkylation of nudeosomes also increasedthe enzymatic release of 3-MeA as well as decreased the sedimentationcoefficient. AD of these experiments indicate a limited availabilityof 3-MeA residues to the glycosylase and suggest that some conformationalchange must occur in vivo for complete repair.     

Induction of the DNA repair enzymes uracil DNA glycosylase and 3-methyladenine DNA glycosylase in regenerating rat liver

The capacity of eukaryotic cells to modulate the activitiesof DNA repair enzymes during cell proliferation was examined.Using regenerating rat liver as a model system, the specificactivities of the DNA repair enzymes uracil DNA glycosylaseand 3-methyladenine DNA glycosylase were determined at specificintervals after partial hepatectomy. The induction of DNA replicationand the stimulation of DNA polymerase were also measured inorder to relate changes in the potential for DNA repair to thoseobserved for DNA replication. As measured in nuclear extracts,the specific activities of both the uracil DNA glycosylase andthe 3-methyladenine DNA glycosylase were increased in regeneratingrat liver reaching maximal levels 18–24 h after partialhepatectomy. The specific activity of each DNA repair enzymereturned to basal levels by 48 h after the hepatectomy. No increasein either enzyme activity was observed in sham operated controls.The products of the reactions were identified as 3-methyladenineor as uracil by high pressure liquid chromatography or by gelfiltration on Sephadex G-10. The 2–3 fold increases inthe specific activity observed for each nuclear DNA repair enzymewas comparable to the 2.7 fold increase observed for DNA polymeraseactivity. The stimulation of DNA repair enzymes in regeneratingrat liver is a further suggestion that eukaryotic cells activelyregulate excision repair pathways in the defined pattern ofgene expression observed during the eukaryotic cell cycle.     

The activity of 3-methyladenine DNA glycosylase in animal tissues in relation to carcinogenesis

3-Methyladenine is one of the major products formed by reactionof a large number of environmental methylating agents with DNAin vivo and in vitro. In spite of the rapid spontaneous depurinationof this base an enzyme, 3-methyladenine DNA glycosylase, hasbeen shown to catalyse its excision. The relevance of this enzymein carcinogenesis induced by alkylating agents was studied.Acute or chronic treatment of rats with diethylnitrosamine orwith N-acetylaminofluorene caused a slight increase in glycosylaseactivity in liver. Experiments with liver regenerating afterpartial hepatectomy showed a similar Increase to occur at thetime of DNA replication. It could be that the increase foundafter treatment with carcinogens was related to the accompanyingincrease in cell replication, rather than being the result ofa specific induction by the carcinogen. Glycosylase activitywas found to be higher in the liver of the rabbit and cat thanin rat or hamster liver. Organ differences (liver, kidney andbrain of the rabbit) were smaller than the species differencesfound for enzyme activity in liver.     

Release of chloroethyl ethyl sulfide-modified DNA bases by bacterial 3-methyladenine-DNA glycosylases I and II

Treatment with chloroethyl ethyl sulfide introduces the followingmodified bases into DNA: 7-ethylthioethylguanine, 3-ethyithioethyladenine,and O⁶-ethylthioethylguanine. Using the ethylthioethylated basesas models for DNA modifications involving relatively bulky alkylgroups, we have investigated the release of these bases by Escherichiacoli 3-methyladenine-DNA glycosylases I and II. 3-Methyladenine-DNAglycosylase I releases only 3-ethylthioethyladenine from chloroethylethyl sulfide-modified DNA, but does so at a rate which exceedsthe rate of release of 3-methyladenine (m³A) from methyl nitrosourea-modifiedDNA under these conditions. 3-Methyladenine-DNA glycosylaseII releases both 3-ethylthioethyl-adenine and 7-ethylthioethylguanineat rates approximating or exceeding the rate of release of m³Afrom methylnitrosourea-modified DNA. We conclude that theseglycosylases may offer some protection against the toxicityof agents which introduce bulky groups into E.coli DNA.     

Influence of DNA structure on hypoxanthine and 1,N(6)-ethenoadenine removal by murine 3-methyladenine DNA glycosylase

3-Methyladenine DNA glycosylases initiate base excision repair by flipping the nucleotide bearing the target base out of double-stranded DNA into an active site pocket for glycosylic bond cleavage and base release. Substrate bases for the murine 3-methyladenine DNA glycosylase (other than 3-methyladenine) include hypoxanthine and 1,N(6)-ethenoadenine, two mutagenic adducts formed by both endogenous and exogenous agents. Using double-stranded DNA oligonucleotides containing damaged bases at specific sites, we studied the relative removal rates for these two adducts when located in different sequence contexts. One of the sequence contexts was an A:T tract, chosen because DNA secondary structure is known to change along the length of this tract, due to a progressive narrowing of the minor groove. Here we report that removal rates for hypoxanthine, but not for 1,N(6)-ethenoadenine, are dramatically affected by its location within the A:T tract. In addition, the removal rates of hypoxanthine and 1,N(6)-ethenoadenine when paired opposite thymine or cytosine were examined, and in each sequence context hypoxanthine removal decreased by at least 20-fold when paired opposite cytosine versus thymine. In contrast, 1, N(6)-ethenoadenine removal was unaffected by the identity of the opposing pyrimidine. We conclude that the removal of certain bases by the mouse 3-methyladenine DNA glycosylase can be modulated by both adjacent and opposing sequence contexts. The influence of DNA sequence context upon DNA repair rates, such as those described here, may contribute to the creation of mutational hot spots in mammalian cells.     

Excision of imidazole ring-opened N7-hydroxyethylguanine from chloroethylnitrosourea-treated DNA by Escherichia coli formamidopyrimidine-DNA glycosylase.

Alkylkation of the N7 of guanine residues in DNA favours the opening of the imidazole ring, yielding a formamidopyrimidine (Fapy). This Fapy residue blocks DNA replication and is actively excised by a DNA glycosylase. We have cloned and sequenced the Escherichia coli gene responsible for synthesis of the enzyme, which has also been purified to homogeneity. It was found to have associated apurinic/apyrimidinic (AP) lyase activity, nicking DNA at AP sites. Chloroethylnitrosoureas are used in cancer chemotherapy. The lesions induced in DNA by these compounds, including N7-chloro- and hydroxyethylguanine, are excised by E. coli 3-methyladenine DNA glycosylase II, and we report that the corresponding imidazole ring-opened forms are repaired by Fapy-DNA glycosylase. Human cells have the counterpart to these enzymes, which could contribute to the repair of these lesions during chemotherapy.     

Antitumour imidazotetrazines XXVIII 3-methyladenine DNA glycosylase activity in cell lines sensitive and resistant to temozolomide.

The ability of extracts of a range of cell lines to release methylated bases from a DNA substrate, which had been modified with [3H]dimethyl sulphate, has been compared in cell lines with differing sensitivity to the cytotoxic drug, temozolomide. High performance liquid chromatography profiles of the bases released by these extracts showed that the activity was specific for 3-methyladenine. There was little variation in the level of 3-methyladenine-DNA glycosylase between the different cell lines despite a 40-fold difference in sensitivity to temozolomide and no correlation with the level of O6-alkylguanine-DNA alkyltransferase.     

Prophylaxis of oxidative DNA damage by formamidopyrimidine-DNA glycosylase

Lying at the gas-exchange interface, lung epithelia may be at risk of oxidation-induced mutagenesis. Further, inflammation processes possibly consequent on smoking liberate reactive oxygen species that multiply the carcinogenic effects of tobacco. DNA repair mechanisms play a major role in counteracting the deleterious effects of oxidative DNA damage. Some studies find positive associations between lung cancer and variations in the human 8-oxoguanine DNA glycosylase (hOGG1) gene that encodes a major DNA glycosylase for oxidized lesions with sluggish kinetics properties. The bacterial homologue formamidopyrimidine-DNA glycosylase (FPG) is 80-fold faster than hOGG1 in repairing mutagenic oxidative lesions. Cell-culture studies have shown that FPG can be expressed in mammalian cells, where it accelerates DNA repair and abates mutagenicity of a wide range of DNA-damaging agents. Prophylaxis of oxidative DNA damage and mutation could be achieved in lung epithelia and other tissues of at-risk individuals by expression of the FPG protein. Currently available vehicles for this peculiar type of gene therapy are briefly surveyed.     

Sequence specificity of DNA repair by Escherichia coli Fpg protein.

The sequence specificity of guanine methylation in DNA by N-methyl-N-nitrosourea and the subsequent repair of ring-opened N7-methylguanine was studied using oligonucleotides of defined sequence. It was found that the methylation of TAGGGGCCCCTA was less than 2-fold greater than that occurring in TAGAGATCTCTA or TATGTGCACATA and 6-fold greater than in TACGCGCGCGTA. This is consistent with the concept that guanine methylation is least when the 5' preceding base is a pyrimidine and greatest when the 5' base is another guanine. These dodecamers were used to study repair by the Escherichia coli Fpg protein (formamidopyrimidine-DNA glycosylase) after the 7-methyl-guanine present in them was converted to the ring-opened form by alkaline treatment. The repair of ring-opened 7-methylguanine was much faster in self-complementary double-stranded 12mer substrates and was twice as rapid at 37 degrees C in TAGGGGCCCCTA compared with TACGCGCGCGTA. However, at 15 degrees C, the relative rates were reversed since TACGCGCGCGTA was repaired at the same rate as at 37 degrees C, whereas the repair of TAGGGGCCCCTA was much slower at 15 degrees C. The repair of TAGGGGCCCCTA at 37 degrees C was also much faster than the repair of TAGAGATCTCTA and was slightly more rapid than repair of TATGTGCACATA. Ligation of the dodecamer substrates to form 24mers or 36mers slightly reduced the initial rates of repair but did not abolish these differences. These results indicate that under physiological conditions, the Fpg protein is more active against adducts in guanine-rich regions and such regions may be the most likely sites of adduct formation at the N7-position of guanine which can then give rise to derivatives attacked by this enzyme.     

3T3 NIH murine fibroblasts and B78 murine melanoma cells expressing the Escherichia coli N3-methyladenine-DNA glycosylase I do not become resistant to alkylating agents

The role of alkylation of the N3 position of adenine in thecytotoxicity of alkylating agents in mammalian cells is stillundefined. By co-transfecting NIH3T3 murine fibroblast and murineB78 H1 melanoma cells with pSG5tag and pSV2neo, we obtainedclones expressing the mRNA of the bacterial tag gene codingfor N3-methyladenine-DNA glycosylase I (Gly I), which specificallyrepairs N3-methyladenine. The levels of Gly I were 400 timeshigher in NIH3T3 pSG5tag (clone 3.9.4.) and 12-33 times higherin B78 HI tag clones (2A4, 2A6, 2C3 and 2D1) than in the respectivecontrol cells. The sensitivity to alkylating agents was evaluatedin tag-expressing cells in comparison with pSG5, pSV2neo co-transfectedcontrol cells. As regards the cytotoxic activity of methylatingagents (N-methylnitrosourea, N-methyl-N-nitro-N'-nitrosoguanidine,dimethylsulfate and temozolomide) and other alkylators withdifferent structure and different interactions with DNA suchas CC-1065 and FCE-24517 (minor groove binders known to bindto A3 of adenine), 4-[bis(2-chloroethyl)amino]-L-phenylalanineand cis-diamino-dichloroplatinum II, cytotoxicity was the samefor tag-expressing and non-expressing cells. These results suggestthat the increased expression of N3-methyladenine-DNA glycosylaseis not necessarily a crucial mechanism for the resistance ofcells to alkylating agents.     

Gene expression caused by alkylating agents and cis-diamminedichloroplatinum(II) in Escherichia coli

Previous work has demonstrated heterogeneous effects of methylating agents on induction of DNA damage inducible genes in Escherichia coli. These studies employed E. coli mutants that have fusions of the lac operon to genes induced by treatment with sublethal levels of alkylating agents. These mutants were selected from random insertions of the Mu-dl (Apr lac) phage by screening for induction of beta-galactosidase activity in the presence of methylmethanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine. The current report extends these findings by analyzing gene expression caused by mechlorethamine, chloroethylnitrosoureas and cis-diamminedichloroplatinum(II) (cis-DDP). The results demonstrate heterogeneous effects by these agents on gene expression. While 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea induces alkA, other nitrosoureas, mechlorethamine, and cis-DDP do not cause expression of this gene. Further, while all nitrosoureas caused expression of aidC, mechlorethamine and cis-DDP did not. Lastly, cis-DDP caused marked expression of a sulA fusion mutant while not inducing any of the other E. coli fusion mutants.     

Excision of the imidazole ring-opened form of N-2-aminofluorene-C(8)-guanine adduct in poly(dG-dC) by Escherichia coli formamidopyrimidine-DNA glycosylase

A polynucleotide containing N-(deoxyguanosine-8-yl)-2-aminofluorene residues (dGuo-C8-AF) was obtained by treatment of poly(dG-dC) with [3H]ring-N-hydroxy-2-amino-fluorene. This substrate was further treated under alkaline conditions to convert dGuo-C8-AF residues into their imidazole ring-opened derivative or 1-[6-(2,5-diamino-4-oxo-pyrimidinyl-N-6-deoxyribose]-3-(2-fluorenyl++ +)urea (iro-dGuo-C8-AF). The ring-opening of 50% of the dGuo-C8-AF residues occurs in 24 h at 37 degrees C in the presence of 0.1 N NaOH. This modified polynucleotide was used as substrate for the homogeneous formamidopyrimidine-DNA glycosylase (Fapy-DNA glycosyase) of Escherichia coli. Analysis of the reaction products shows that Fapy-DNA glycosylase releases the imidazole ring-opened derivative (iro-G-C8-AF). In contrast the primary adduct (G-C8-AF) is not removed. These results show that the imidazole ring-opened form of guanine residue modified at the C8 position by a bulky adduct is a substrate for the formamidopyrimidine-DNA glycosylase of E. coli. These observations show that the formamidopyrimidine-DNA glycosylase has a broad substrate specificity including imidazole ring-opened purines either modified at N7 or C8 positions in DNA. Therefore, the Fapy-DNA glycosylase might be involved in the repair of minor lesions induced by many chemical carcinogens.     

Analysis of uracil DNA glycosylase in human colorectal cancer

Uracil DNA glycosylase (UDG) is responsible for the removal of uracil present in DNA after cytosine deamination or misincorporation during replication. Colorectal cancer is widely treated with 5-FU, which leads to thymidylate synthase inhibition; this accounts for increased dUTP intracellular pools and subsequent uracil incorporation into DNA. Uracil misincorporation has also been implicated in the link between folate deficiency and colorectal cancer risk. As there is no information on UDG in colorectal cancer, this study characterized UDG activity and protein expression in a panel of 20 colorectal tumors and 6 colorectal cell lines. UDG activity in colorectal tissue is widely variable and it is statistically higher in tumor tissue (P=0.013) compared to normal bowel. Tumor versus normal activity ratios ranged from 0.49 to 2.2 (median 1.13). Among the six colorectal cell lines tested, UDG activity varied from 40 to 68 units and was markedly (1.7-fold) higher than in tumor tissue (P<0.0001). In both colorectal tissues and cell lines, UDG was expressed as both 29 kDa and 35 kDa forms. Total protein expression varied 3.2-fold in cell lines; variability was also found between patients and between normal and tumoral tissue for the same patient. This study demonstrates UDG protein and functional activity in human colorectal tumors and cell lines. The high tumor:normal tissue ratio supports further interest in base excision repair, through UDG, as a potential source of fluoropyrimidine resistance in colorectal cancer.     

Detection of DNA damage by Escherichia coli UvrB-binding competition assay is limited by the stability of the UvrB-DNA complex

To investigate the use of UvrB-binding to detect DNA damage, mobility shift gel electrophoresis was used to detect binding of UvrB protein to a 136 bp DNA fragment that was randomly adducted with aflatoxin B1 8,9- epoxide and end-labelled with 32P. After polyacrylamide gel electrophoresis, the shifted band that contained DNA bound by UvrB was quantified as a percentage of total radioactive substrate DNA. This method was applied to analyse plasmid DNA that was adducted with various DNA modifying agents in vitro. These adducts competed for UvrB- binding to the labelled substrate. By competing for UvrB-binding with 10 ng of plasmid DNA that was adducted with known levels of aflatoxin B1, 2-amino-3-methylimidazo[4,5-f]quinoline, or benzo[a]pyrene diol epoxide, UvrB competition could be quantified for DNA adducted with between one adduct in 10(2) and one adduct in 10(5) normal nucleotides. However, plasmid DNA exposed to N-methyl-N-nitrosourea or methylene blue + visible light, did not compete for UvrB-binding, even though the presence of UvrABC sensitive sites were confirmed on this DNA by a UvrABC incision assay. Mono-adducted 96-bp DNA substrates, which contained an internal 32P-label and either a single apurinic site, aflatoxin B1-guanine adduct, O6-methylguanine, 8-oxo-deoxyguanosine or non-adducted guanine, were also used as substrates for UvrA- and UvrB- binding to examine the stability of UvrB-DNA complexes with specific adducts. Under similar conditions used for the competition assay, significant UvrB-binding was seen only for the aflatoxin adducted substrate. These results suggest that stability of UvrB-binding varies greatly between bulky and non-bulky adducts. It was also found that rat liver DNA from untreated rats inhibited UvrB-binding to the substrate DNA in the competition assay, to a degree that was equivalent to competition with plasmid adducted at one adduct in 10(3) normal nucleotides.      

DNA sequence specificity of doxorubicin-induced mutational damage in uvrB- Escherichia coli.

In the absence of excision repair, doxorubicin caused a striking (41-fold) increase in the frequency of large deletion mutations extending from the lac operator (lacO) into the lac repressor gene (lacI) of Escherichia coli. In contrast, there was only a 2-fold increase in the frequency of small deletions despite a 3-fold increase in overall mutation frequency. The 5'-endpoints of doxorubicin-induced lacO and lacI/lacO deletions occurred at the DNA sequence 5'-pyTAA or 5'-AATpy (where py is pyrmidine) (16%), at runs of purines or pyrimidines (41%) and adjacent to 5'-dGdC or 5'-dCdG doublets (34%). Ninety % (27 of 30) of the doxorubicin-induced deletions involving the region of the lacO palindrome had 3'-endpoints within the palindrome sequence as compared with 40% (4 of 10) spontaneous deletions in an untreated set. Doxorubicin-induced single base substitutions were highly focused at one site (4 of 6) in the i-d region of lacI, in contrast to the spontaneous distribution of point mutations, where 16 mutants were recovered at 12 different sites. An increased frequency (3-fold) of highly focused base substitutions was also observed at 2 sites in the lac operator region (at lacO +6, which is a transition "hotspot" in the spontaneous spectra of both wild type and uvrB- organisms and at the adjacent +5 site). Notably, the frequency of 1- and 2-base frameshifts did not increase in the doxorubicin-induced spectrum, relative to the spontaneous mutation spectrum. These in vivo observations in E. coli suggest that in the absence of excision repair, doxorubicin causes highly focused deletions and base substitutions. These mutations occur adjacent to DNA sequences identified in previous in vitro studies as preferential sites of doxorubicin binding.     

5-Fluorouracil incorporated into DNA is excised by the Smug1 DNA glycosylase to reduce drug cytotoxicity

5-Fluorouracil (FU) has been widely used for more than four decades in the treatment of a range of common cancers. The fluorine-substituted uracil analogue is converted to several active metabolites but the mechanism of cytotoxicity has remained unclear. In a widely cited but unsubstantiated model, FU is thought to kill cells via the inhibition of thymidylate synthase and increased use of dUTP in place of TTP during DNA replication, with subsequent excision of high levels of uracil causing the fragmentation of newly synthesized DNA. Using gene-targeted cell lines defective in one or both of the two mammalian uracil-DNA glycosylase repair enzymes, we were able to test this model of FU cytotoxicity. Here, we show that incorporation of FU itself into DNA has been previously underestimated and is a predominant cause of cytotoxicity. FU readily becomes incorporated into the DNA of drug-treated cells, and accumulation of FU in the genome, rather than uracil excision, is correlated with FU cytotoxicity in mammalian cells. Furthermore, the Smug1, but not the Ung, uracil-DNA glycosylase excises FU from DNA and protects against cell killing. The data provides a clearer understanding of the action of FU, suggesting predictive biomarkers of drug response and a mechanism for acquired resistance in tumors.     

The Escherichia coli MutS DNA mismatch binding protein specifically binds O6-methylguanine DNA lesions

DNA mismatch repair defects in certain cell types confer resistanceto the cytotoxic effects of alkylating agents, suggesting thata normally functioning DNA mismatch repair pathway can actuallymediate alkylation-induced cell death. In eukaryotic cells thisphenomenon is only observed in cells lacking adequate DNA methyltransferasefor the repair of O⁶-methylguanine (O⁶MeG) DNA lesions. It hasbeen proposed that O⁶MeG may act as a substrate for DNA mismatchrepair when paired with cytosine and when mispaired with thymineand that repeated futile DNA mismatch repair at O⁶MeG DNA lesionsis cytotoxic. Here we show that the Escherichia coli MutS DNAmismatch repair binding protein does indeed bind specificallyto O⁶MeG DNA lesions. In contrast, MutS does not bind DNA containinganother O-alkylated base, namely O⁴-methylthymine, or anotherkind of modified guanine, namely 8-oxoguanine. These resultsprovide direct biochemical evidence for the involvement of DNAmismatch repair in specifically processing O⁶MeG DNA lesions.