Increased A:T-->C:G mutations in the mutT strain upon 8-hydroxy-dGTP treatment: direct evidence for MutT involvement in the prevention of mutations by oxidized dGTP

The Escherichia coli MutT protein hydrolyzes 8-hydroxy-dGTP (8-OH-dGTP) in vitro, and mutT gene deficiencies cause increased spontaneous A:T-->C:G mutations. However, no direct evidence exists for enhanced mutagenicity of 8-OH-dGTP in mutT cells. In this study, 8-OH-dGTP was introduced into wild type and mutT E. coli cells, and mutations of a chromosomal gene were monitored. 8-OH-dGTP induced mutations of the rpoB gene, the degree of the mutation induction in the mutT strain being approximately 6-fold higher than that in the wild type strain. On the other hand, 2-hydroxy-dATP, which is not a substrate of the MutT protein, increased the mutation to similar degrees in the two strains. These results constitute the first evidence that the MutT protein suppresses mutation by 8-OH-dGTP in vivo.     

Involvement of specialized DNA polymerases in mutagenesis by 8-hydroxy-dGTP in human cells

The mutagenicity of an oxidized form of dGTP, 8-hydroxy-2′-deoxyguanosine 5′-triphosphate (8-OH-dGTP), was examined using human 293T cells. Shuttle plasmid DNA containing the supF gene was first transfected into the cells, and then 8-OH-dGTP was introduced by means of osmotic pressure. The DNAs replicated in the cells were recovered and then transfected into Escherichia coli. 8-OH-dGTP induced A:T → C:G substitution mutations in the cells. The knock-downs of DNA polymerases η and ζ, and REV1 by siRNAs reduced the A:T → C:G substitution mutations, suggesting that these DNA polymerases are involved in the misincorporation of 8-OH-dGTP opposite A in human cells. In contrast, the knock-down of DNA polymerase ι did not affect the 8-OH-dGTP-induced mutations. The decrease in the induced mutation frequency was more evident by double knock-downs of DNA pols η plus ζ and REV1 plus DNA pol ζ (but not by that of DNA pol η plus REV1), suggesting that REV1-DNA pol η and DNA pol ζ work in different steps. These results indicate that specialized DNA polymerases are involved in the mutagenesis induced by the oxidized dGTP.     

Modulation of oxidative mutagenesis and carcinogenesis by polymorphic forms of human DNA repair enzymes

Chromosome DNA is continuously exposed to various endogenous and exogenous mutagens. Among them, oxidation is one of the most common threats to genetic stability, and multiple DNA repair enzymes protect chromosome DNA from the oxidative damage. In Escherichia coli, three repair enzymes synergistically reduce the mutagenicity of oxidized base 8-hydroxy-guanine (8-OH-G). MutM DNA glycosylase excises 8-OH-G from 8-OH-G:C pairs in DNA and MutY DNA glycosylase removes adenine incorporated opposite template 8-OH-G during DNA replication. MutT hydrolyzes 8-OH-dGTP to 8-OH-dGMP in dNTP pool, thereby reducing the chance of misincorporation of 8-OH-dGTP by DNA polymerases. Simultaneous inactivation of MutM and MutY dramatically increases the frequency of spontaneous G:C to T:A mutations, and the deficiency of MutT leads to the enhancement of T:A to G:C transversions more than 1000-fold over the control level. In humans, the functional homologues of MutM, MutY and MutT, i.e., OGG1, MUTYH (MYH) and MTH1, contribute to the protection of genomic DNA from oxidative stress. Interestingly, several polymorphic forms of these proteins exist in human populations, and some of them are suggested to be associated with cancer susceptibility. Here, we review the polymorphic forms of OGG1, MUTYH and MTH1 involved in repair of 8-OH-G and 8-OH-dGTP, and discuss the significance of the polymorphisms in the maintenance of genomic integrity. We also summarize the polymorphic forms of human DNA polymerase eta, which may be involved in damage tolerance and mutagenesis induced by oxidative stress.     

2-Hydroxy-2'-deoxyadenosine 5'-triphosphate enhances A.T --> C.G mutations caused by 8-hydroxy-2'-deoxyguanosine 5'-triphosphate by suppressing its degradation upon replication in a HeLa extract

The coexistence effects of multiple kinds of oxidized deoxyribonucleotides were examined using an SV40 origin-dependent in vitro replication system with a HeLa extract. Oxidized dGTP and dATP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP) and 2-hydroxy-2'-deoxyadenosine 5'-triphosphate (2-OH-dATP), were used in this study. The mutation frequency synergistically increased when the two oxidized deoxyribonucleotides were together in the reaction. 2-OH-dATP enhanced the mutagenicity of 8-OH-dGTP, since the induced mutations were A.T --> C.G transversions. The contribution of the highly error-prone DNA polymerase eta was unlikely, since similar results were observed with an XP-V cell extract. The possible involvement of 2-hydroxyadenine in the complementary (template) strand was excluded on the basis of experiments using plasmids containing 2-hydroxyadenine as templates in the reactions with 8-OH-dGTP. 2-OH-dATP suppressed hydrolysis of 8-OH-dGTP, suggesting that the inhibition of the MTH1 protein played the major role in the enhancement. These results highlight the importance of specific hydrolysis of 8-OH-dGTP for the suppression of its induced mutation.     

Specificity of mutations induced by incorporation of oxidized dNTPs into DNA by human DNA polymerase eta

Aberrant oxidation is a property of many tumor cells. Oxidation of DNA precursors, i.e., deoxynucleotide triphosphates (dNTPs), as well as DNA is a major cause of genome instability. Here, we report that human DNA polymerase eta (h Poleta) incorporates oxidized dNTPs, i.e., 2-hydroxy-2'-deoxyadenosine 5'-triphosphate (2-OH-dATP) and 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP), into DNA in an erroneous and efficient manner, thereby inducing various types of mutations during in vitro gap-filling DNA synthesis. When 2-OH-dATP was present at a concentration equal to those of the four normal dNTPs in the reaction mixture, DNA synthesis by h Poleta enhanced the frequency of G-to-T transversions eight-fold higher than that of the transversions in control where only the normal dNTPs were present. When 8-OH-dGTP was present at an equimolar concentration to the normal dNTPs, it enhanced the frequency of A-to-C transversions 17-fold higher than the control. It also increased the frequency of C-to-A transversions about two-fold. These results suggest that h Poleta incorporates 2-OH-dATP opposite template G and incorporates 8-OH-dGTP opposite template A and slightly opposite template C during DNA synthesis. Besides base substitutions, h Poleta enhanced the frequency of single-base frameshifts and deletions with the size of more than 100 base pairs when 8-OH-dGTP was present in the reaction mixture. Since h Poleta is present in replication foci even without exogenous DNA damage, we suggest that h Poleta may be involved in induction of various types of mutations through the erroneous and efficient incorporation of oxidized dNTPs into DNA in human cells.     

Mutagenicity of secondary oxidation products of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-hydroxy-2'- deoxyguanosine 5'-triphosphate)

8-Oxo-7,8-dihydroguanine (8-hydroxyguanine) is oxidized more easily than normal nucleobases, which can produce spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). These secondary oxidation products of 8-oxo-7,8-dihydroguanine are highly mutagenic when formed within DNA. To evaluate the mutagenicity of the corresponding oxidation products of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-hydroxy-2'- deoxyguanosine 5'-triphosphate) in the nucleotide pool, Escherichia coli cells deficient in the mutT gene were treated with H(2)O(2), and the induced mutations were analyzed. Moreover, the 2'-deoxyriboside 5'-triphosphate derivatives of Sp and Gh were also introduced into competent E. coli cells. The H(2)O(2) treatment of mutT E. coli cells resulted in increase of G:C → T:A and A:T → T:A mutations. However, the incorporation of exogenous Sp and Gh 2'-deoxyribonucleotides did not significantly increase the mutation frequency. These results suggested that the oxidation product(s) of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate induces G:C → T:A and A:T → T:A mutations, and that the 2'-deoxyriboside 5'-triphosphate derivatives of Sp and Gh exhibit quite weak mutagenicity, in contrast to the bases in DNA.     

Efficient and erroneous incorporation of oxidized DNA precursors by human DNA polymerase eta

Altered oxidative metabolism is a property of many tumor cells. Oxidation of DNA precursors, i.e., dNTP pool, as well as DNA is a major source of mutagenesis and carcinogenesis. Here, we report the remarkable nature of human DNA polymerase eta that incorporates oxidized dNTPs into a nascent DNA strand in an efficient and erroneous manner. The polymerase almost exclusively incorporated 8-hydroxy-dGTP (8-OH-dGTP) opposite template adenine (A) at 60% efficiency of normal dTTP incorporation, and incorporated 2-hydroxy-dATP (2-OH-dATP) opposite template thymine (T), guanine (G), or cytosine (C) at substantial rates. The synthetic primers having 8-hydroxy-G paired with template A or 2-hydroxy-A paired with template T, G, or C at the termini were efficiently extended. In contrast, human DNA polymerase iota incorporated 8-OH-dGTP opposite template A with much lower efficiency and did not incorporate 2-OH-dATP opposite any of the template bases. It did not extend the primers having the oxidized bases at the termini either. We propose that human DNA polymerase eta may participate in oxidative mutagenesis through the efficient and erroneous incorporation of oxidized dNTPs during DNA synthesis.     

Base excision repair enzyme endonuclease III suppresses mutagenesis caused by 8-hydroxy-dGTP

To examine whether base excision repair suppresses mutations induced by oxidized deoxyribonucleotide 5'-triphosphates in the nucleotide pool, 8-hydroxy-dGTP (8-OH-dGTP) and 2-hydroxy-dATP were introduced into Escherichia coli strains deficient in endonucleases III (Nth) and VIII (Nei) and MutY, and mutations in the chromosomal rpoB gene were analyzed. The spontaneous rpoB mutant frequency was also examined in mutT/nth and mutT/nei strains, to assess the influence on the mutations induced by the endogenous 8-OH-dGTP accumulated in the mutT mutant. The mutations induced by exogenous 2-hydroxy-dATP were similar in all of the strains tested. Exogenous 8-OH-dGTP increased the rpoB mutant frequency more efficiently in the nth strain than that in the wild-type strain. The spontaneous mutant frequency in the mutT/nth strain was 2-fold higher than that in the mutT strain. These results suggest that E. coli endonuclease III also acts as a defense against the mutations caused by 8-OH-dGTP in the nucleotide pool.     

Mutations induced by 5-formyl-2'-deoxyuridine in Escherichia coli include base substitutions that can arise from mispairs of 5-formyluracil with guanine, cytosine and thymine

5-Formyluracil (5-foU) is a major oxidation product of thymine formed in yields comparable to that of 8-oxoguanine in DNA by ionizing radiation. Whereas the mutagenic effects of 8-oxoguanine are well understood, the investigation of the biological implications of 5-foU has so far been limited. Here we demonstrate that 5-formyl-2'-deoxyuridine (5-fodUrd) supplied to the growth medium of Escherichia coli induces several base substitutions at different frequencies at position 461 in the lacZ gene in the following order: A.T-->G.C>G.C-->A.T>G.C-->T.A>A.T-->T.A>A.T-->C.G. No induction of G.C-->C.G transversions was observed. It is inferred that 5-fodUrd will be incorporated into the DNA during cell growth, forming mispairs with guanine, cytosine and thymine during replication. It, thus, appears that cell growth in the presence of 5-fodUrd may represent a good model for elucidating the cellular effects of 5-foU residues in DNA.     

Specificity of spontaneous mutations induced in mutA mutator cells

Escherichia coli cells expressing the mutA allele of a glyV (glycine tRNA) gene express a strong mutator phenotype. The mutA allele differs from the wild type glyV gene by a base substitution in the anticodon such that the resulting tRNA misreads certain aspartate codons as glycine, resulting in random, low-level Asp-->Gly substitutions in proteins. Subsequent work showed that many types of mistranslation can lead to a very similar phenotype, named TSM for translational stress-induced mutagenesis. Here, we have determined the specificity of forward mutations occurring in the lacI gene in mutA cells as well as in wild type cells. Our results show that in comparison to wild type cells, base substitutions are elevated 23-fold in mutA cells, as against a eight-fold increase in insertions and a five-fold increase in deletions. Among base substitutions, transitions are elevated 13-fold, with both G:C-->A:T and A:T-->G:C mutations showing roughly similar increases. Transversions are elevated 35-fold, with G:C-->T:A, G:C-->C:G and A:T-->C:G elevated 28-, 13- and 27-fold, respectively. A:T-->T:A mutations increase a striking 348-fold over parental cells, with most occurring at two hotspot sequences that share the G:C-rich sequence 5'-CCGCGTGG. The increase in transversion mutations is similar to that observed in cells defective for dnaQ, the gene encoding the proofreading function of DNA polymerase III. In particular, the relative proportions and sites of occurrence of A:T-->T:A transversions are similar in mutA and mutD5 (an allele of dnaQ) cells. Interestingly, transversions are also the predominant base substitutions induced in dnaE173 cells in which a missense mutation in the alpha subunit of polymerase III abolishes proofreading without affecting the 3'-->5' exonuclease activity of the epsilon subunit.     

Mutagenesis of uracil-DNA glycosylase deficient mutants of the extremely thermophilic eubacterium Thermus thermophilus

Thermus thermophilus is an extremely thermophilic, aerobic, and gram-negative eubacterium that grows optimally at 70-75 degrees C, pH 7.5. In extremely high temperature environment, DNA damages in cells occur at a much higher frequency in thermophiles than mesophiles such as E. coli. When temperature rises, the deamination of cytosine residues in double-strand DNA is expected to increase greatly. T. thermophilus HB27 has two putative uracil-DNA glycosylase genes (udgA and udgB). Expression level of udgA gene was 2-3 times higher than that of udgB at 70, 74, and 78 degrees C when it was monitored by beta-glucosidase reporter assay. We developed hisD(3110), hisD(3113), hisD(3115), and hisD(174) marker allele that can specifically detect G:C-->A:T, C:G-->A:T, T:A-->A:T, and A:T-->G:C base-substitutions, respectively, by His(+) reverse mutations. We then disrupted udgA and udgB by thermostable kanamycin-resistant gene (htk) or pyrE gene insertion in each hisD background, and their spontaneous His(+) reversion frequencies were compared. A udgA,B double mutant showed a pronounced increase in G:C-->A:T reversion frequency compared with each single udg mutant, udgA or udgB. Estimated mutation rates of the udgA,B mutant cultured at 60, 70, and 78 degrees C were about 2, 12, and 117 His(+)/10(8)/generation, respectively. At 70 degrees C culture, increased ratio of the mutation rate compared with the udg(+) strain was 12-fold in udgA, 3-fold in udgB, and 56-fold in udgA,B mutant. On the other hand, no difference was observed in other mutations of C:G-->A:T, T:A-->A:T, and A:T-->G:C between udgA,B double mutant and the parent udg(+) strain. The present results indicated that gene products of udgB as well as udgA functioned in vivo to remove uracil in DNA and prevent G:C-->A:T transition mutations.     

Genetic effects of oxidative DNA damage: comparative mutagenesis of 7,8-dihydro-8-oxoguanine and 7,8-dihydro-8-oxoadenine in Escherichia coli.

A single 7,8-dihydro-8-oxoguanine (G8-OXO; 8-hydroxyguanine) adduct in the lacZ alpha gene of bacteriophage M13 DNA induces a targeted G-->T transversion after replication in Escherichia coli (Biochemistry, 29, 7024-7031 (1990)). This mutation is thought to be due to the facile formation during DNA synthesis of a G8-OXO.base pair, where G8-OXO is in the syn conformation about the deoxyglycosyl bond. A related modified purine, 7,8-dihydro-8-oxoadenine (A8-OXO; 8-hydroxyadenine), is an abundant product found in irradiated and oxidized DNAs. Similar to G8-OXO, as a mononucleoside A8-OXO assumes the syn conformation. This work has assessed the relative mutagenicities of A8-OXO and G8-OXO in the same experimental system. A deoxypentanucleotide containing A8-OXO [d(GCT-A8-OXOG)] was synthesized. After 5'-phosphorylation with [gamma-32P] ATP, the oligomer was ligated into a duplex M13mp19-derived genome at a unique NheI restriction site. Genomes containing either A8-OXO (at position 6275, [+] strand) or G8-OXO (position 6276) were denatured with heat and introduced into E.coli DL7 cells. Analysis of phage DNA from mutant plaques obtained by plating immediately after transformation (infective centers assay) revealed that G8-OXO induced G-->T transversions at an apparent frequency of approximately 0.3%. The frequency and spectrum of mutations observed in DNA sequences derived from 172 mutant plaques arising from the A8-OXO-modified DNA were almost indistiguishable from those generated from transfection of an adenine-containing control genome. We conclude that A8-OXO is at least an order of magnitude less mutagenic than G8-OXO in E.coli cells with normal DNA repair capabilities.     

Mutational specificity of mice defective in the MTH1 and/or the MSH2 genes

Oxidative damage of nucleotides within DNA or precursor pools caused by oxygen radicals is thought to play an important role in spontaneous mutagenesis, as well as carcinogenesis and aging. In particular, 8-oxodGTP and 2-OHdATP are potent mutagenic substrate for DNA synthesis. Mammalian MTH1 catalyzes hydrolysis of these mutagenic substrates, suggesting that it functions to prevent mutagenesis caused by these oxidized nucleotides. We have established MTH1(-/-) mice lacking the 8-oxodGTPase activity, which were shown to be susceptible to lung, liver and stomach cancers. To examine in vivo mutation events due to the MTH1-deficiency, a reporter gene, rpsL of Escherichia coli, was introduced into MTH1(-/-) mice. Interestingly, the net frequency of rpsL(-) forward mutants showed no apparent increase in MTH1(-/-) mice as compared to MTH1(+/+) mice. However, we found differences between these two genotypes in the class- and site-distributions of the rpsL(-) mutations recovered from the mice. Unlike MutT-deficient E. coli showing 1000-fold higher frequency of A:T-->C:G transversion than the wild type cells, an increase in frequency of A:T-->C:G transversion was not evident in MTH1 nullizygous mice. Nevertheless, the frequency of single-base frameshifts at mononucleotide runs was 5.7-fold higher in spleens of MTH1(-/-) mice than in those of wild type mice. Since the elevated incidence of single-base frameshifts at mononucleotide runs is a hallmark of the defect in MSH2-dependent mismatch repair system, this weak site-specific mutator effect of MTH1(-/-) mice could be attributed to a partial sequestration of the mismatch repair function that may act to correct mispairs with the oxidized nucleotides. Consistent with this hypothesis, a significant increase in the frequency of G:C-->T:A transversions was observed with MTH1(-/-) MSH2(-/-) mice over MSH2(-/-) mice alone. These results suggest a possible involvement of multiple anti-mutagenic pathways, including the MTH1 protein and other repair system(s), in mutagenesis caused by the oxidized nucleotides.     

Induction of T --> G and T --> A transversions by 5-formyluracil in mammalian cells.

Oxidatively damaged thymine, 5-formyluracil (5-fU), was incorporated into a predetermined site of double-stranded shuttle vectors. The nucleotide sequences in which the modified base was incorporated were 5'-CFTAAG-3' and 5'-CTFAAG-3' (F represents 5-fU), the recognition site for the restriction enzyme AflII (5'-CTTAAG-3'). The 5-fU was incorporated into a template strand of either the leading or lagging strand of DNA replication. The modified DNAs were transfected into simian COS-7 cells, and the DNAs replicated in the cells were recovered and were analyzed after the second transfection into Escherichia coli. The 5-fU did not block DNA replication in mammalian cells. The 5-fU residues were weakly mutagenic, and their mutation frequencies in double-stranded vectors were 0.01-0.04%. The T --> G and T --> A transversions were the mutations found most frequently, suggesting the formation of 5-fU.C and 5-fU.T base pairs, respectively. This is the first report that clearly shows the induction of transversion mutations by an oxidized pyrimidine base in DNA in mammalian cells.     

The interaction of the Escherichia coli mutD and mutT pathways in the prevention of A:T-->C:G transversions.

The Escherichia coli mutT mutator allele produces high frequencies of exclusively A:T-->C:G transversions. This is thought to be caused by a failure to prevent or remove A:G mispairs during DNA replication. The mutD5 mutator allele maps to the dnaQ locus which encodes the epsilon subunit of the DNA polymerase III holoenzyme. This subunit provides 3'-->5' exonuclease, proofreading, activity for removing mispaired nucleotides at the 3' end of the newly synthesized DNA strand. mutD5 has an altered epsilon resulting in reduced levels of proofreading and subsequent high mutation frequencies for all base-pair substitutions. We have analyzed the interaction between mutD5 and mutT-induced A:T-->C:G transversions by measuring reversion frequencies in mutD5 and mutT single mutator strains and mutD5mutT double mutator strains using the well-characterized trpA58 and trpA88 alleles. We find that the double mutator strains produce more A:T-->C:G substitutions than would be expected from simple additivity of the single mutator strains. We interpret this to mean that the two systems, at least in part, do act together to prevent the same mutational intermediate from producing A:T-->C:G transversions. It is estimated that over 90% of the mutT-induced A:G mispairs are corrected by proofreading at the trpA58 site while only about 30% are corrected at trpA88. Reversion frequencies in the mutD5mutT double mutator strains indicate A:G misincorporations occur about 100 x more frequently at trpA58 than at the trpA88 site. Using these and other data we also provide estimations of the fidelity contributions for mutT editing, proofreading and methyl-directed mismatch repair at the two trpA sites for both transversions and the transition that could be scored. In the case of A:T-->C:G transversions, both mutT editing and proofreading make major contributions in error reduction with mismatch repair playing a small or no role at all. For the A:T-->G:C transition, proofreading and mismatch repair were both important in preventing mutations while no contribution was observed for mutT editing.     

The oxidized forms of dATP are substrates for the human MutT homologue, the hMTH1 protein.

The possibility that Escherichia coli MutT and human MTH1 (hMTH1) hydrolyze oxidized DNA precursors other than 8-hydroxy-dGTP (8-OH-dGTP) was investigated. We report here that hMTH1 hydrolyzed 2-hydroxy-dATP (2-OH-dATP) and 8-hydroxy-dATP (8-OH-dATP), oxidized forms of dATP, but not (R)-8,5'-cyclo-dATP, 5-hydroxy-dCTP, and 5-formyl-dUTP. The kinetic parameters indicated that 2-OH-dATP was hydrolyzed more efficiently and with higher affinity than 8-OH-dGTP. 8-OH-dATP was hydrolyzed as efficiently as 8-OH-dGTP. The preferential hydrolysis of 2-OH-dATP over 8-OH-dGTP was observed at all of the pH values tested (pH 7.2 to pH 8.8). In particular, a 5-fold difference in the hydrolysis efficiencies for 2-OH-dATP over 8-OH-dGTP was found at pH 7.2. However, E. coli MutT had no hydrolysis activity for either 2-OH-dATP or 8-OH-dATP. Thus, E. coli MutT is an imperfect counterpart for hMTH1. Furthermore, we found that 2-hydroxy-dADP and 8-hydroxy-dGDP competitively inhibited both the 2-OH-dATP hydrolase and 8-OH-dGTP hydrolase activities of hMTH1. The inhibitory effects of 2-hydroxy-dADP were 3-fold stronger than those of 8-hydroxy-dGDP. These results suggest that the three damaged nucleotides share the same recognition site of hMTH1 and that it is a more important sanitization enzyme than expected thus far.     

Interactions among the Escherichia coli mutT,mutM, and mutY damage prevention pathways

We have investigated in detail the interactions between the Escherichia coli mutT, mutM, and mutY error-prevention systems. Jointly, these systems protect the cell against the effects of the oxidative stress product, 8-oxoguanine (8-oxoG), a base analog with ambiguous base-pairing properties, pairing with either A or C during DNA synthesis. mutT mutator strains display a specific increase in A.T-->C.G transversions, while mutM and mutY mutator strains show specific G.C-->T.A increases. To study in more detail the in vivo processing of the various mutational intermediates leading to A.T-->C.G and G.C-->T.A transversions, we analyzed defined A.T-->C.G and G.C-->T.A events in strains containing all possible combinations of these mutator alleles. We report three major findings. First, we do not find evidence that the mutT allele significantly increases G.C-->T.A transversions in either mut(+), mutM, mutY or mutMmutY backgrounds. We interpret this result to indicate that incorporation of 8-oxodGTP opposite template C may not be frequent relative to incorporation opposite template A. Second, we show that mutT-induced A.T-->C.G transversions are significantly reduced in strains carrying mutY and mutMmutY deficiencies suggesting that 8-oxoG, when present in DNA, preferentially mispairs with dATP. Third, the mutY and mutMmutY deficiencies also decrease A.T-->C.G transversions in the mutT(+) background, suggesting that, even in the presence of functional MutT protein, A.T-->C.G transversions may still result from 8-oxodGTP misincorporation.     

Endonuclease V protects Escherichia coli against specific mutations caused by nitrous acid

Endonuclease V (deoxyinosine 3'-endonuclease) of Escherichia coli K-12 is a putative DNA repair enzyme that cleaves DNA's containing hypoxanthine, uracil, or mismatched bases. An endonuclease V (nfi) mutation was tested for specific mutator effects on a battery of trp and lac mutant alleles. No marked differences were seen in frequencies of spontaneous reversion. However, when nfi mutants were treated with nitrous acid at a level that was not noticeably mutagenic for nfi(+) strains, they displayed a high frequency of A:T-->G:C, and G:C-->A:T transition mutations. Nitrous acid can deaminate guanine in DNA to xanthine, cytosine to uracil, and adenine to hypoxanthine. The nitrous acid-induced A:T-->G:C transitions were consistent with a role for endonuclease V in the repair of deaminated adenine residues. A confirmatory finding was that the mutagenesis was depressed at a locus containing N(6)-methyladenine, which is known to be relatively resistant to nitrosative deamination. An alkA mutation did not significantly enhance the frequency of A:T-->G:C mutations in an nfi mutant, even though AlkA (3-methyladenine-DNA glycosylase II) has hypoxanthine-DNA glycosylase activity. The nfi mutants also displayed high frequencies of nitrous acid-induced G:C-->A:T transitions. These mutations could not be explained by cytosine deamination because an ung (uracil-DNA N-glycosylase) mutant was not similarly affected. However, these findings are consistent with a role for endonuclease V in the removal of deaminated guanine, i.e., xanthine, from DNA. The results suggest that endonuclease V helps to protect the cell against the mutagenic effects of nitrosative deamination.     

The importance of distinct metabolites of N-nitrosodiethylamine for its in vivo mutagenic specificity

Although N-nitrosodiethylamine (NDEA) is a potent carcinogen in rodents and a probable human carcinogen, little attempts were made to characterize its mutation spectrum in higher eukaryotes. We have compared forward mutation frequencies at multiple (700) loci with the mutational spectrum induced at the vermilion gene of Drosophila, after exposure of post- and pre-meiotic male germ cells to NDEA. Among 30 vermilion mutants collected from post-meiotic stages were 12 G:C-->A:T transitions (40%), 8 A:T-->T:A transversions (27%), and 4 structural rearrangements (13%). The remainder were three A:T-->G:C transitions, two G:C-->C:G transversions and one G:C-->T:A transversion. The results show that although NDEA induces predominantly transitions (40% G:C-->A:T and 10% A:T-->G:C), the frequencies of transversions (37%, of which 27% of A:T-->T:A transversions) and especially of rearrangements (13%) are remarkably high. This mutation spectrum differs significantly from that produced by the direct-ethylating agent N-ethylnitrosourea (ENU), although the relative distribution of ethylated DNA adducts is similar for both carcinogens. These differences, in particular the occurrence of rearrangements, are most likely the result of the requirement of NDEA for bioactivation. Since all four rearrangements were collected from non-metabolizing spermatozoa (or late spermatids), it is hypothesized that they derived from acetaldehyde, a stable metabolite of NDEA. Due to its cytotoxicity, attempts to isolate vermilion mutants from NDEA-exposed pre-meiotic cells were largely unsuccessful, because only two mutants (one A:T-->G:C transition and one 1bp insertion) were collected from those stages. Our results show that NDEA is capable of generating carcinogenic lesions other than base pair substitutions.