In Escherichia coli themutT gene is one of several that acts to minimize mutagenesis by reactive oxygen species. The bacterial MutT protein and its mammalian homolog have been shown to catalyze in vitrothe hydrolysis of the oxidized deoxyguanosine nucleotide, 8-oxo-dGTP, to its corresponding monophosphate. Thus, the protein is thought to “sanitize” the nucleotide pool by ridding the cell of a nucleotide whose incorporation into DNA would be intensely mutagenic. However, because others have shown mutT mutations to be mutagenic under some conditions of anaerobic growth, and have shown 8-oxo-dGTP to be a poor DNA polymerase substrate, there is reason to question this model. We have devised an assay for 8-oxo-dGTP in bacterial extracts. Using this assay, which involves reversed-phase high-performance liquid chromatography and electrochemical detection, we have been unable to detect 8-oxo-dGTP in extracts of three different mutTmutants of E. coli, even after growth of the bacteria in the presence of hydrogen peroxide. Our estimated upper limit for 8-oxo-dGTP content of these bacteria is about 200 molecules/cell, corresponding to a concentration of about 0.34 μm. When 8-oxo-dGTP was added at 0.34 μm to an in vitro DNA replication system primed with a DNA template that permits scoring of replication errors and with the four normal dNTPs at their estimated intracellular concentrations, there was no detectable effect upon the frequency of replication errors. These findings lead us to question the conclusion that 8-oxo-dGTP is the most significant physiological substrate for the MutT protein. In Escherichia coli themutT gene is one of several that acts to minimize mutagenesis by reactive oxygen species. The bacterial MutT protein and its mammalian homolog have been shown to catalyze in vitrothe hydrolysis of the oxidized deoxyguanosine nucleotide, 8-oxo-dGTP, to its corresponding monophosphate. Thus, the protein is thought to “sanitize” the nucleotide pool by ridding the cell of a nucleotide whose incorporation into DNA would be intensely mutagenic. However, because others have shown mutT mutations to be mutagenic under some conditions of anaerobic growth, and have shown 8-oxo-dGTP to be a poor DNA polymerase substrate, there is reason to question this model. We have devised an assay for 8-oxo-dGTP in bacterial extracts. Using this assay, which involves reversed-phase high-performance liquid chromatography and electrochemical detection, we have been unable to detect 8-oxo-dGTP in extracts of three different mutTmutants of E. coli, even after growth of the bacteria in the presence of hydrogen peroxide. Our estimated upper limit for 8-oxo-dGTP content of these bacteria is about 200 molecules/cell, corresponding to a concentration of about 0.34 μm. When 8-oxo-dGTP was added at 0.34 μm to an in vitro DNA replication system primed with a DNA template that permits scoring of replication errors and with the four normal dNTPs at their estimated intracellular concentrations, there was no detectable effect upon the frequency of replication errors. These findings lead us to question the conclusion that 8-oxo-dGTP is the most significant physiological substrate for the MutT protein. 7,8-dihydro-8-oxoguanine 8-oxodeoxyguanosine triphosphate deoxyribonucleoside 5′-triphosphate high-performance liquid chromatography 5-bromo-4-chloro-3-indoyl-β-d-galactoside human immunodeficiency virus The action of reactive oxygen species upon cells stimulates mutagenesis in large part by increasing the abundance in DNA of the oxidized guanine derivative, 7,8-dihydro-8-oxoguanine (8-oxoG).1 This base efficiently pairs with adenine, leading, if unrepaired, to a transversion mutation (1Michaels M.L. Miller J.H. J. Bacteriol. 1992; 174: 6321-6325Crossref PubMed Scopus (608) Google Scholar). In Escherichia coli the products of three genes play particularly prominent roles in counteracting this genotoxicity, and all three have homologs in mammalian cells. Two of these genes, mutM and mutY, encode DNA glycosylases, and their actions initiate base excision repair processes at sites occupied by oxoG (1Michaels M.L. Miller J.H. J. Bacteriol. 1992; 174: 6321-6325Crossref PubMed Scopus (608) Google Scholar). The third gene, mutT, encodes a nucleotidase, which cleaves dGTP to dGMP and pyrophosphate (2Bhatnagar S.K. Bullions L.C. Bessman M.J. J. Biol. Chem. 1991; 266: 9050-9054Abstract Full Text PDF PubMed Google Scholar) but which Maki and Sekiguchi (3Maki H. Sekiguchi M. Nature. 1992; 355: 273-275Crossref PubMed Scopus (782) Google Scholar) showed to have much lowerKm for the oxidized dGTP derivative, 8-oxo-dGTP. This finding plus subsequent publications from the same laboratory (4Mo J-Y. Maki H. Sekiguchi M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11021-11025Crossref PubMed Scopus (297) Google Scholar, 5Sakumi K. Furuichi M. Tsuzuki T. Kakuma T. Kawabata S-i. Maki H. Sekiguchi M. J. Biol. Chem. 1993; 268: 23524-23530Abstract Full Text PDF PubMed Google Scholar, 6Hayakawa H. Taketomi A. Sakumi K. Kuwano M. Sekiguchi M. Biochemistry. 1995; 34: 89-95Crossref PubMed Scopus (149) Google Scholar, 7Tajiri T. Maki H. Sekiguchi M. Mutation Res. 1995; 336: 257-267Crossref PubMed Scopus (316) Google Scholar, 8Kakuma T. Nishida J.-i. Tsuzuki T. Sekiguchi M. J. Biol. Chem. 1995; 270: 25942-25948Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 9Hayakawa H. Hofer A. Thelander L. Kitajima S. Cai Y. Oshiro S. Yakushiji H. Nakabeppu Y. Kuwano M. Sekiguchi M. Biochemistry. 1999; 38: 3610-3614Crossref PubMed Scopus (125) Google Scholar) support the concept that the action of the MutT protein is to “sanitize” the nucleotide pool by removing from cells a damaged nucleotide that, if incorporated into DNA, would be strongly mutagenic. In agreement with this model, several investigators (10Cheng K.C. Cahill D.S. Kasai H. Nishimura S. Loeb L.A. J. Biol. Chem. 1992; 267: 166-172Abstract Full Text PDF PubMed Google Scholar, 11Pavlov Y.I. Minnick D.T. Izuta S. Kunkel T.A. Biochemistry. 1994; 33: 4695-4701Crossref PubMed Scopus (138) Google Scholar, 12Minnick D.T. Pavlov Y.I. Kunkel T.A. Nucleic Acids Res. 1994; 22: 5658-5664Crossref PubMed Scopus (37) Google Scholar) have shown that addition of 8-oxo-dGTP to an in vitro DNA replication system in which replication errors could be scored as mutations stimulated replication errors that were shown by sequence analysis to be transversions.The human homolog of MutT, hMTH1, has been expressed in mutTmutant E. coli and shown to suppress the mutator phenotype (5Sakumi K. Furuichi M. Tsuzuki T. Kakuma T. Kawabata S-i. Maki H. Sekiguchi M. J. Biol. Chem. 1993; 268: 23524-23530Abstract Full Text PDF PubMed Google Scholar, 8Kakuma T. Nishida J.-i. Tsuzuki T. Sekiguchi M. J. Biol. Chem. 1995; 270: 25942-25948Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Thus, both bacterial and mammalian proteins are thought to play the same role in sanitizing the nucleotide pool. The bacterial and human enzymes differ somewhat in substrate specificity; hMTH1 acts upon two oxidized dATP derivatives as well as upon 8-oxo-dGTP (13Fujikawa K. Kamiya H. Yakushiji H. Fujii Y. Nakabeppu Y. Kasai H. J. Biol. Chem. 1999; 274: 18201-18205Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar), and the bacterial enzyme acts upon 8-oxo-GTP, the ribonucleotide analog of 8-oxo-dGTP (14Taddei F. Hayakawa H. Bouton M.-F. Cirinesi A.-M. Matic I. Sekiguchi M. Radman M. Science. 1997; 278: 128-131Crossref PubMed Scopus (246) Google Scholar).Although most published data support the nucleotide pool cleansing role for MutT in ridding the cell of 8-oxo-dGTP, two puzzling observations have been made. First, Fowler et al. (15Fowler R.G. Erickson J.A. Isbell R.J. J. Bacteriol. 1994; 176: 7727-7729Crossref PubMed Google Scholar) showed that under certain growth conditions for E. coli the mutTmutator phenotype is expressed even when cells are grown anaerobically, and this is under conditions where no appreciable dGTP oxidation should be taking place. Second, Einolf et al. (16Einolf H.J. Schnetz-Boutaud N. Guengerich F.P. Biochemistry. 1998; 37: 13300-13312Crossref PubMed Scopus (97) Google Scholar, 17Einolf H.J. Guengerich F.P. J. Biol. Chem. 2001; 276: 3764-3771Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) showed 8-oxo-dGTP to be a poor substrate for several DNA polymerases when compared with dGTP. For four different polymerases (16Einolf H.J. Schnetz-Boutaud N. Guengerich F.P. Biochemistry. 1998; 37: 13300-13312Crossref PubMed Scopus (97) Google Scholar), thekcat/Km was ∼105-fold higher for dGTP:C than for either 8-oxo-dGTP:A or 8-oxo-dGTP:C, suggesting that 8-oxo-dGTP levels in cells would have to be quite high in order for it to substitute for dGTP to a significant extent and hence to play a significant role in mutagenesis. Note that in studies with the in vitro DNA replication systems (10Cheng K.C. Cahill D.S. Kasai H. Nishimura S. Loeb L.A. J. Biol. Chem. 1992; 267: 166-172Abstract Full Text PDF PubMed Google Scholar, 11Pavlov Y.I. Minnick D.T. Izuta S. Kunkel T.A. Biochemistry. 1994; 33: 4695-4701Crossref PubMed Scopus (138) Google Scholar, 12Minnick D.T. Pavlov Y.I. Kunkel T.A. Nucleic Acids Res. 1994; 22: 5658-5664Crossref PubMed Scopus (37) Google Scholar), 8-oxo-dGTP was present at concentrations equal to or greater than those of dGTP, conditions that are unlikely to hold in living cells.To better understand the role of 8-oxo-dGTP in mutagenesis and the mechanism by which MutT minimizes the effects of oxidative DNA damage, it seemed important for intracellular levels of 8-oxo-dGTP to be measured directly under various conditions. For example, we would expect to see higher levels of 8-oxo-dGTP in mutT mutator mutant bacteria than in wild-type cells and lower levels in anaerobic than in aerobic growth conditions. Developing a protocol for extracting and analyzing the 8-oxo-dGTP pool in E. coli and carrying out such analyses were the goals of this investigation.DISCUSSIONWe have developed a separation and analytical system that allows the detection of as little as 6 pmol of 8-oxo-dGTP in a bacterial extract containing the normal nucleotides at their ordinary concentrations. However, when extracts of wild-type and mutTbacteria were compared, we were not able to detect the oxidized nucleotide even at this level in either wild-type or mutant cell extracts, whether or not the bacteria had been oxidatively stressed beforehand by exposure to hydrogen peroxide. To be sure, our electrochemical detection profiles did reveal the presence in mutant cell extracts of at least one electrochemically active substance (Fig.3, C and D) that is distinct from 8-oxo-dGTP. Identification of this material might provide a clue to understanding the mutator phenotype of mutT mutants. However, at present we do not even know whether the unknown material is a nucleotide; preliminary data indicate that it is not an oxidized derivative of one of the standard ribo- or deoxyribonucleoside triphosphates. Clearly, identification of this material, which is apparently present in quite small amounts, might provide a clue to additional biochemical functions of the MutT protein, and that is one of the directions our future research will take.Our observations with an in vitro replication error detection system are consistent with the results of Einolf et al. (16Einolf H.J. Schnetz-Boutaud N. Guengerich F.P. Biochemistry. 1998; 37: 13300-13312Crossref PubMed Scopus (97) Google Scholar), who reported that 8-oxo-dGTP is a very poor substrate, as compared with dGTP, for four different DNA polymerases: E. coli polymerases I and II, T7 polymerase, and HIV reverse transcriptase. Three of those polymerases do not play their primary roles in chromosomal DNA replication. However, more recently Einolf and Guengerich (17Einolf H.J. Guengerich F.P. J. Biol. Chem. 2001; 276: 3764-3771Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) observed the same result when they analyzed 8-oxo-dGTP as a substrate for a mammalian replicative polymerase, DNA polymerase δ, in the presence of proliferating cell nuclear antigen. Ourin vitro experiments were done with a human cell extract system (22Roberts J.D. Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7064-7068Crossref PubMed Scopus (70) Google Scholar) that carries out semiconservative replication initiating at normal replication origins. Using a mutational target in which most or all substitution mutations can be detected, we saw no significant increase over the control mutant fraction when replication was carried out in the presence of standard 8-oxo-dGTP at the experimentally determined upper limit of its intracellular concentration. These observations, coupled with expression of themutT mutator phenotype under anaerobic growth conditions (15Fowler R.G. Erickson J.A. Isbell R.J. J. Bacteriol. 1994; 176: 7727-7729Crossref PubMed Google Scholar), suggest that the mechanism by which the MutT protein counteracts oxidative mutagenesis needs to be re-evaluated. The action of reactive oxygen species upon cells stimulates mutagenesis in large part by increasing the abundance in DNA of the oxidized guanine derivative, 7,8-dihydro-8-oxoguanine (8-oxoG).1 This base efficiently pairs with adenine, leading, if unrepaired, to a transversion mutation (1Michaels M.L. Miller J.H. J. Bacteriol. 1992; 174: 6321-6325Crossref PubMed Scopus (608) Google Scholar). In Escherichia coli the products of three genes play particularly prominent roles in counteracting this genotoxicity, and all three have homologs in mammalian cells. Two of these genes, mutM and mutY, encode DNA glycosylases, and their actions initiate base excision repair processes at sites occupied by oxoG (1Michaels M.L. Miller J.H. J. Bacteriol. 1992; 174: 6321-6325Crossref PubMed Scopus (608) Google Scholar). The third gene, mutT, encodes a nucleotidase, which cleaves dGTP to dGMP and pyrophosphate (2Bhatnagar S.K. Bullions L.C. Bessman M.J. J. Biol. Chem. 1991; 266: 9050-9054Abstract Full Text PDF PubMed Google Scholar) but which Maki and Sekiguchi (3Maki H. Sekiguchi M. Nature. 1992; 355: 273-275Crossref PubMed Scopus (782) Google Scholar) showed to have much lowerKm for the oxidized dGTP derivative, 8-oxo-dGTP. This finding plus subsequent publications from the same laboratory (4Mo J-Y. Maki H. Sekiguchi M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11021-11025Crossref PubMed Scopus (297) Google Scholar, 5Sakumi K. Furuichi M. Tsuzuki T. Kakuma T. Kawabata S-i. Maki H. Sekiguchi M. J. Biol. Chem. 1993; 268: 23524-23530Abstract Full Text PDF PubMed Google Scholar, 6Hayakawa H. Taketomi A. Sakumi K. Kuwano M. Sekiguchi M. Biochemistry. 1995; 34: 89-95Crossref PubMed Scopus (149) Google Scholar, 7Tajiri T. Maki H. Sekiguchi M. Mutation Res. 1995; 336: 257-267Crossref PubMed Scopus (316) Google Scholar, 8Kakuma T. Nishida J.-i. Tsuzuki T. Sekiguchi M. J. Biol. Chem. 1995; 270: 25942-25948Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 9Hayakawa H. Hofer A. Thelander L. Kitajima S. Cai Y. Oshiro S. Yakushiji H. Nakabeppu Y. Kuwano M. Sekiguchi M. Biochemistry. 1999; 38: 3610-3614Crossref PubMed Scopus (125) Google Scholar) support the concept that the action of the MutT protein is to “sanitize” the nucleotide pool by removing from cells a damaged nucleotide that, if incorporated into DNA, would be strongly mutagenic. In agreement with this model, several investigators (10Cheng K.C. Cahill D.S. Kasai H. Nishimura S. Loeb L.A. J. Biol. Chem. 1992; 267: 166-172Abstract Full Text PDF PubMed Google Scholar, 11Pavlov Y.I. Minnick D.T. Izuta S. Kunkel T.A. Biochemistry. 1994; 33: 4695-4701Crossref PubMed Scopus (138) Google Scholar, 12Minnick D.T. Pavlov Y.I. Kunkel T.A. Nucleic Acids Res. 1994; 22: 5658-5664Crossref PubMed Scopus (37) Google Scholar) have shown that addition of 8-oxo-dGTP to an in vitro DNA replication system in which replication errors could be scored as mutations stimulated replication errors that were shown by sequence analysis to be transversions. The human homolog of MutT, hMTH1, has been expressed in mutTmutant E. coli and shown to suppress the mutator phenotype (5Sakumi K. Furuichi M. Tsuzuki T. Kakuma T. Kawabata S-i. Maki H. Sekiguchi M. J. Biol. Chem. 1993; 268: 23524-23530Abstract Full Text PDF PubMed Google Scholar, 8Kakuma T. Nishida J.-i. Tsuzuki T. Sekiguchi M. J. Biol. Chem. 1995; 270: 25942-25948Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Thus, both bacterial and mammalian proteins are thought to play the same role in sanitizing the nucleotide pool. The bacterial and human enzymes differ somewhat in substrate specificity; hMTH1 acts upon two oxidized dATP derivatives as well as upon 8-oxo-dGTP (13Fujikawa K. Kamiya H. Yakushiji H. Fujii Y. Nakabeppu Y. Kasai H. J. Biol. Chem. 1999; 274: 18201-18205Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar), and the bacterial enzyme acts upon 8-oxo-GTP, the ribonucleotide analog of 8-oxo-dGTP (14Taddei F. Hayakawa H. Bouton M.-F. Cirinesi A.-M. Matic I. Sekiguchi M. Radman M. Science. 1997; 278: 128-131Crossref PubMed Scopus (246) Google Scholar). Although most published data support the nucleotide pool cleansing role for MutT in ridding the cell of 8-oxo-dGTP, two puzzling observations have been made. First, Fowler et al. (15Fowler R.G. Erickson J.A. Isbell R.J. J. Bacteriol. 1994; 176: 7727-7729Crossref PubMed Google Scholar) showed that under certain growth conditions for E. coli the mutTmutator phenotype is expressed even when cells are grown anaerobically, and this is under conditions where no appreciable dGTP oxidation should be taking place. Second, Einolf et al. (16Einolf H.J. Schnetz-Boutaud N. Guengerich F.P. Biochemistry. 1998; 37: 13300-13312Crossref PubMed Scopus (97) Google Scholar, 17Einolf H.J. Guengerich F.P. J. Biol. Chem. 2001; 276: 3764-3771Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) showed 8-oxo-dGTP to be a poor substrate for several DNA polymerases when compared with dGTP. For four different polymerases (16Einolf H.J. Schnetz-Boutaud N. Guengerich F.P. Biochemistry. 1998; 37: 13300-13312Crossref PubMed Scopus (97) Google Scholar), thekcat/Km was ∼105-fold higher for dGTP:C than for either 8-oxo-dGTP:A or 8-oxo-dGTP:C, suggesting that 8-oxo-dGTP levels in cells would have to be quite high in order for it to substitute for dGTP to a significant extent and hence to play a significant role in mutagenesis. Note that in studies with the in vitro DNA replication systems (10Cheng K.C. Cahill D.S. Kasai H. Nishimura S. Loeb L.A. J. Biol. Chem. 1992; 267: 166-172Abstract Full Text PDF PubMed Google Scholar, 11Pavlov Y.I. Minnick D.T. Izuta S. Kunkel T.A. Biochemistry. 1994; 33: 4695-4701Crossref PubMed Scopus (138) Google Scholar, 12Minnick D.T. Pavlov Y.I. Kunkel T.A. Nucleic Acids Res. 1994; 22: 5658-5664Crossref PubMed Scopus (37) Google Scholar), 8-oxo-dGTP was present at concentrations equal to or greater than those of dGTP, conditions that are unlikely to hold in living cells. To better understand the role of 8-oxo-dGTP in mutagenesis and the mechanism by which MutT minimizes the effects of oxidative DNA damage, it seemed important for intracellular levels of 8-oxo-dGTP to be measured directly under various conditions. For example, we would expect to see higher levels of 8-oxo-dGTP in mutT mutator mutant bacteria than in wild-type cells and lower levels in anaerobic than in aerobic growth conditions. Developing a protocol for extracting and analyzing the 8-oxo-dGTP pool in E. coli and carrying out such analyses were the goals of this investigation. DISCUSSIONWe have developed a separation and analytical system that allows the detection of as little as 6 pmol of 8-oxo-dGTP in a bacterial extract containing the normal nucleotides at their ordinary concentrations. However, when extracts of wild-type and mutTbacteria were compared, we were not able to detect the oxidized nucleotide even at this level in either wild-type or mutant cell extracts, whether or not the bacteria had been oxidatively stressed beforehand by exposure to hydrogen peroxide. To be sure, our electrochemical detection profiles did reveal the presence in mutant cell extracts of at least one electrochemically active substance (Fig.3, C and D) that is distinct from 8-oxo-dGTP. Identification of this material might provide a clue to understanding the mutator phenotype of mutT mutants. However, at present we do not even know whether the unknown material is a nucleotide; preliminary data indicate that it is not an oxidized derivative of one of the standard ribo- or deoxyribonucleoside triphosphates. Clearly, identification of this material, which is apparently present in quite small amounts, might provide a clue to additional biochemical functions of the MutT protein, and that is one of the directions our future research will take.Our observations with an in vitro replication error detection system are consistent with the results of Einolf et al. (16Einolf H.J. Schnetz-Boutaud N. Guengerich F.P. Biochemistry. 1998; 37: 13300-13312Crossref PubMed Scopus (97) Google Scholar), who reported that 8-oxo-dGTP is a very poor substrate, as compared with dGTP, for four different DNA polymerases: E. coli polymerases I and II, T7 polymerase, and HIV reverse transcriptase. Three of those polymerases do not play their primary roles in chromosomal DNA replication. However, more recently Einolf and Guengerich (17Einolf H.J. Guengerich F.P. J. Biol. Chem. 2001; 276: 3764-3771Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) observed the same result when they analyzed 8-oxo-dGTP as a substrate for a mammalian replicative polymerase, DNA polymerase δ, in the presence of proliferating cell nuclear antigen. Ourin vitro experiments were done with a human cell extract system (22Roberts J.D. Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7064-7068Crossref PubMed Scopus (70) Google Scholar) that carries out semiconservative replication initiating at normal replication origins. Using a mutational target in which most or all substitution mutations can be detected, we saw no significant increase over the control mutant fraction when replication was carried out in the presence of standard 8-oxo-dGTP at the experimentally determined upper limit of its intracellular concentration. These observations, coupled with expression of themutT mutator phenotype under anaerobic growth conditions (15Fowler R.G. Erickson J.A. Isbell R.J. J. Bacteriol. 1994; 176: 7727-7729Crossref PubMed Google Scholar), suggest that the mechanism by which the MutT protein counteracts oxidative mutagenesis needs to be re-evaluated. We have developed a separation and analytical system that allows the detection of as little as 6 pmol of 8-oxo-dGTP in a bacterial extract containing the normal nucleotides at their ordinary concentrations. However, when extracts of wild-type and mutTbacteria were compared, we were not able to detect the oxidized nucleotide even at this level in either wild-type or mutant cell extracts, whether or not the bacteria had been oxidatively stressed beforehand by exposure to hydrogen peroxide. To be sure, our electrochemical detection profiles did reveal the presence in mutant cell extracts of at least one electrochemically active substance (Fig.3, C and D) that is distinct from 8-oxo-dGTP. Identification of this material might provide a clue to understanding the mutator phenotype of mutT mutants. However, at present we do not even know whether the unknown material is a nucleotide; preliminary data indicate that it is not an oxidized derivative of one of the standard ribo- or deoxyribonucleoside triphosphates. Clearly, identification of this material, which is apparently present in quite small amounts, might provide a clue to additional biochemical functions of the MutT protein, and that is one of the directions our future research will take. Our observations with an in vitro replication error detection system are consistent with the results of Einolf et al. (16Einolf H.J. Schnetz-Boutaud N. Guengerich F.P. Biochemistry. 1998; 37: 13300-13312Crossref PubMed Scopus (97) Google Scholar), who reported that 8-oxo-dGTP is a very poor substrate, as compared with dGTP, for four different DNA polymerases: E. coli polymerases I and II, T7 polymerase, and HIV reverse transcriptase. Three of those polymerases do not play their primary roles in chromosomal DNA replication. However, more recently Einolf and Guengerich (17Einolf H.J. Guengerich F.P. J. Biol. Chem. 2001; 276: 3764-3771Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) observed the same result when they analyzed 8-oxo-dGTP as a substrate for a mammalian replicative polymerase, DNA polymerase δ, in the presence of proliferating cell nuclear antigen. Ourin vitro experiments were done with a human cell extract system (22Roberts J.D. Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7064-7068Crossref PubMed Scopus (70) Google Scholar) that carries out semiconservative replication initiating at normal replication origins. Using a mutational target in which most or all substitution mutations can be detected, we saw no significant increase over the control mutant fraction when replication was carried out in the presence of standard 8-oxo-dGTP at the experimentally determined upper limit of its intracellular concentration. These observations, coupled with expression of themutT mutator phenotype under anaerobic growth conditions (15Fowler R.G. Erickson J.A. Isbell R.J. J. Bacteriol. 1994; 176: 7727-7729Crossref PubMed Google Scholar), suggest that the mechanism by which the MutT protein counteracts oxidative mutagenesis needs to be re-evaluated. We thank our colleague Dr. William Baird and the Center for Gene Research and Biotechnology for the long-term loan of an HPLC electrochemical detector and a Beckman HPLC unit, respectively, and we thank members of Dr. Baird's laboratory for instructions in use of the electrochemical detector. We thank Stella Martomo of this laboratory for assistance with the in vitroDNA replication reactions. Special thanks are due to Dr. Michael Tassotto for technical support.
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