Escherichia coli mutator (Delta)polA is defective in base mismatch correction: the nature of in vivo DNA replication errors

We constructed a set of Escherichia coli strains containing deletions in genes encoding three SOS polymerases, and defective in MutS and DNA polymerase I (PolI) mismatch repair, and estimated the rate and specificity of spontaneous endogenous tonB(+)-->tonB- mutations. The rate and specificity of mutations in strains proficient or deficient in three SOS polymerases was compared and found that there was no contribution of SOS polymerases to the chromosomal tonB mutations. MutS-deficient strains displayed elevated spontaneous mutation rates, consisting of dominantly minus frameshifts and transitions. Minus frameshifts are dominated by warm spots at run-bases. Among 57 transitions (both G:C-->A:T and A:T-->G:C), 35 occurred at two hotspot sites. PolI-deficient strains possessed an increased rate of deletions and frameshifts, because of a deficiency in postreplicative deletion and frameshift mismatch corrections. Frameshifts in PolI-deficient strains occurred within the entire tonB gene at non-run and run sequences. MutS and PolI double deficiency indicated a synergistic increase in the rate of deletions, frameshifts and transitions. In this case, mutS-specific hotspots for frameshifts and transitions disappeared. The results suggested that, unlike the case previously known pertaining to postreplicative MutS mismatch repair for frameshifts and transitions and PolI mismatch repair for frameshifts and deletions, PolI can recognize and correct transition mismatches. Possible mechanisms for distinct MutS and PolI mismatch repair are discussed. A strain containing deficiencies in three SOS polymerases, MutS mismatch repair and PolI mismatch repair was also constructed. The spectrum of spontaneous mutations in this strain is considered to represent the spectrum of in vivo DNA polymerase III replication errors. The mutation rate of this strain was 219x10(-8), about a 100-fold increase relative to the wild-type strain. Uncorrected polymerase III replication errors were predominantly frameshifts and base substitutions followed by deletions.     

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.     

The roles of Klenow processing and flap processing activities of DNA polymerase I in chromosome instability in Escherichia coli K12 strains.

The sequences of spontaneous mutations occurring in the endogenous tonB gene of Escherichia coli in the DeltapolA and polA107 mutant strains were compared. Five categories of mutations were found: (1) deletions, (2) minus frameshifts, (3) plus frameshifts, (4) duplications, and (5) other mutations. The DeltapolA strain, which is deficient in both Klenow domain and 5' --> 3' exonuclease domain of DNA polymerase I, shows a marked increase in categories 1-4. The polA107 strain, which is deficient in the 5' --> 3' exonuclease domain but proficient in the Klenow domain, shows marked increases in categories 3 and 4 but not in 1 or 2. Previously, we reported that the polA1 strain, which is known to be deficient in the Klenow domain but proficient in the 5' --> 3' exonuclease domain, shows increases in categories 1 and 2 but not in 3 or 4. The 5' --> 3' exonuclease domain of DNA polymerase I is a homolog of the mammalian FEN1 and the yeast RAD27 flap nucleases. We therefore proposed the model that the Klenow domain can process deletion and minus frameshift mismatch in the nascent DNA and that flap nuclease can process plus frameshift and duplication mismatch in the nascent DNA.     

DNA sequence analysis of spontaneous tonB deletion mutations in a polA1 strain of Escherichia coli K12.

By DNA sequence analysis, we have determined a spectrum of 61 spontaneous mutations occurring in the endogenous tonB gene in the polA1 strain of Escherichia coli. The overall mutation frequency was approximately 2.4-fold higher in the polA1 strain and this was attributable to enhanced rates of deletion and frameshift mutations. Among 39 deletions, a hot spot (17 mutations) was detected: a 13-bp deletion presumably directed by a 3-bp repeated sequence at its end points. The remaining 22 were distributed among 19 different mutations either flanked (16/19) or not flanked (3/19) by repeated sequences. Single-base frameshifts accounted for 8 mutations of either repeated (3/8) or nonrepeated (5/8) bases among which 6 were minus one frameshift. In contrast to previous reports, we did not frequently observe a 5'-GTGG-3' sequence in the vicinity of the deletions and frameshifts. The results presented here indicated an anti-deletion and anti-frameshift role for DNA polymerase I.     

Characterization of mutations induced by N-methyl-N'-nitro-N-nitrosoguanidine in an industrial Corynebacterium glutamicum strain

Mutations induced by classical whole-cell mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine (NTG) were determined for all genes of pathways from glucose to L-lysine in an industrial L-lysine producer of Corynebacterium glutamicum. A total of 50 mutations with a genome-wide distribution were identified and characterized for mutational types and mutagenic specificities. Those mutations were all point mutations with single-base substitutions and no deletions, frame shifts, and insertions were found. Among six possible types of base substitutions, the mutations consisted of only two types: 47 G.C-->A.T transitions and three A.T-->G.C transitions with no transversion. The findings indicate a limited repertoire of amino acid substitutions by classical NTG mutagenesis and thus raise a new possibility of further improving industrial strains by optimizing key mutations through PCR-mediated site-directed mutagenesis.     

Specificity of mutational DNA sequence changes induced by X-rays in the cloned Escherichia coli crp gene.

Plasmid DNA carrying the adenosine 3',5'-cyclic monophosphate receptor protein (crp) gene of Escherichia coli was irradiated, in solution, with X-rays, and the mutations produced in the crp gene were assayed by transforming the recipient E. coli cells. Ninety-six mutant clones were isolated, and mutational changes were determined by DNA sequencing. Of the 92 mutations thus detected, 74 represented base substitution mutations and the remaining 18 were frameshifts. The base substitutions included 56 G:C to A:T transitions, 10 G:C to T:A transversions and 7 G:C to C:G transversions. An A:T to G:C transition was found only once, and neither an A:T to T:A nor an A:T to C:G transversion was detected. The frameshift mutations consisted of 11 one-base deletions and 7 one-base insertions. Accordingly, G:C to A:T transition was the predominant type of mutation, which constituted 76% (56/74) of the total base substitutions and 60% (56/92) of all detected mutations. Furthermore, of the 56 transitions, about three-quarters (41 clones) clustered at an identical site, a cytosine residue at the 706 position, demonstrating that this site is a distinct hot spot for X-ray mutagenesis. These results raise the possibility that radiation-induced mutations may not necessarily occur randomly, at least in certain cases.     

Do DNA repair systems affect N4-hydroxycytidine-induced mutagenesis?

It was tested whether mutations induced in E. coli by N4-hydroxycytidine (oh4Cyd): (i) undergo mutation frequency decline (MFD) when synthesis of protein is arrested, and (ii) are influenced by polA1, polA107 or xth mutations. It was also investigated whether oh4Cyd may provoke SOS response and prophage lambda induction. All these processes may involve the action of repair enzymes. It has been shown that none of these processes or repair enzymes affects oh4Cyd-induced mutagenesis.     

The reversion of trp frameshift mutations in mut,polA, lig and dnaE mutant strains of Escherichia coli

Mutator mutations mutL25, mutR34, and mutU4 had similar effects on the reversion of 4 trp frameshift mutations of known sequence. The mutation trpE9777, which resulted from the addition of an A–T base-pair to a run of 5 A–T base-pairs, was most strongly reverted by the 4 mutators. Reversion of trpE9777 was also increased by mutation polA1 (DNA polymerase I) and dnaE486 and dnaE511 (DNA polymerase III). No effect was found with the ligase mutations, lig-4 or lig-ts7. Mutations polAex1 and polA107, both deficient in the 5′ → 3′ exonuclease activity of DNA polymerase I, had different mutator effects; the factor increase in reversion of trpE9777 was 28-fold for polAex1, 6-fold for polA107, and 21-fold for polA1. The trpE9777 mutation is a useful indicator of frameshift mutator activity.     

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.     

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.     

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.     

Frameshift mutagenesis by acridines in wild-type, uvrB and polA strains of Salmonella typhimurium with and without plasmid pKM101

A large range of acridines, including several anilinoacridines which are active as antitumour agents, have been studied for their ability to revert derivatives of Salmonella typhimurium strains carrying the frameshift marker hisC3076. The strains used all carried deep-rough (rfa) mutations, and were either wild-type with respect to DNA-repair capacity or carried uvrB, polA1 or polA3 (amber) mutations. Derivatives with and without the mutation-enhancing N group plasmid pKM101 were also used. 9-Aminoacridine and other acridines appeared similar to the anilinoacridines for the most part, in that frameshift mutagenesis and toxicity appeared to be unaffected by the uvrB mutation or by the presence of plasmid pKM101. Exceptions were ICR191, 3-NO2-acridine and 1- or 3-NO2-anilinoacridine derivatives in which mutagenesis was increased in uvrB strains and also when pKM101 was present. These compounds were slightly more toxic in the uvrB background, but less toxic when pKM101 was present in either the uvrB or wild-type backgrounds. Mutagenesis by most compounds was reduced by the polA1 mutation and virtually eliminated (except in the case of ICR191) by the polA3 mutation. Plasmid pKM101 occasionally enhanced mutagenesis in the polA1 strain, whereas in the polA3 it appeared to have no effect whatsoever. Again, there were no obvious differences in toxicity between Pol+ and Pol- strains.     

REV3 is required for spontaneous but not methylation damage-induced mutagenesis of Saccharomyces cerevisiae cells lacking O6-methylguanine DNA methyltransferase

O6-methylguanine (O6-MeG) DNA methyltransferase (MTase) removes the methyl group from a DNA lesion and directly restores DNA structure. It has been shown previously that bacterial and yeast cells lacking such MTase activity are not only sensitive to killing and mutagenesis by DNA methylating agents, but also exhibit an increased spontaneous mutation rate. In order to understand molecular mechanisms of endogenous DNA alkylation damage and its effects on mutagenesis, we determined the spontaneous mutational spectra of the SUP4-o gene in various Saccharomyces cerevisiae strains. To our surprise, the mgt1 mutant deficient in DNA repair MTase activity exhibited a significant increase in G:C-->C:G transversions instead of the expected G:C-->A:T transition. Its mutational distribution strongly resembles that of the rad52 mutant defective in DNA recombinational repair. The rad52 mutational spectrum has been shown to be dependent on a mutagenesis pathway mediated by REV3. We demonstrate here that the mgt1 mutational spectrum is also REV3-dependent and that the rev3 deletion offsets the increase of the spontaneous mutation rate seen in the mgt1 strains. These results indicate that the eukaryotic mutagenesis pathway is directly involved in cellular processing of endogenous DNA alkylation damage possibly by the translesion bypass of lesions at the cost of G:C-->C:G transversion mutations. However, the rev3 deletion does not affect methylation damage-induced killing and mutagenesis of the mgt1 mutant, suggesting that endogenous alkyl lesions may be different from O6-MeG.     

Absence of strand bias for deletion mutagenesis during chromosomal leading and lagging strand replication in Escherichia coli

Investigations were carried out to determine whether both DNA strands involved in Escherichia coli chromosomal DNA replication are replicated with similar accuracy. Experiments consisted of measuring the forward mutation rate from tonB(+) to tonB(-) in pairs of polA deficient strains in which the chromosomal target gene tonB was oriented in the two possible directions relative to the origin of replication, oriC. Within these pairs, the tonB sequence would be subjected to leading strand replication in one orientation and to lagging strand replication in the other. The most common tonB mutations in the polA1 strain were deletions followed by frameshifts. Among the deletions, a strong hotspot site with a 13-base deletion in the polA1 strains accounted for 18 of the 33 deletions in the one orientation, and 31 of the 58 deletions in the other. The results suggested that the two strands were replicated with equal or similar accuracy for deletion formation.     

An in vivo approach to identifying sequence context of 8-oxoguanine mutagenesis

Base substitution mutations are not distributed randomly in that most are located at a few specific hotspots sites. We have been studying 7,8-dihydro-8-oxoguanine mutagenesis in Escherichia coli in the supF gene carried in a plasmid. Among hotspots, guanine within the 5'-AGA-3' located in the anticodon site was susceptible to the induction of G:C-->T:A transversion. In this study, we constructed variants of the supF gene in which the hotspot 5'-AGA-3' was modified to 5'-AGT-3', 5'-AGG-3' and 5'-AGC-3' to determine the influence of 3' neighboring base on G:C-->T:A mutational activity. Using these variant supF genes propagated in a 7,8-dihydro-8-oxoguanine repair-deficient host, we found that guanine within 5'-AGA-3' and 5'-AGG-3' produce G:C-->T:A, but guanine within 5'-AGT-3' and 5'-AGC-3' reduce the formation of G:C-->T:A. These changes were thus due to the effect of sequence context on the efficiency of mutation formation at the sites of 7,8-dihydro-8-oxoguanine. We also observed a longer range base-pair effect on hotspot formation.     

Powerful mutator activity of the polA1 mutation within the histidine region of Escherichia coli K-12.

We examined 122 spontaneous histidine auxotrophs accumulated in overnight cultures of polA1 strains of Escherichia coli K-12 at approximate frequencies of 10(-3). One hundred and thirteen appeared to be minus frameshifts, and nine appeared to be deletions. Of the frameshift mutations, 109 affected the hisC gene, and 4 affected genes hisD, hisH, hisA, and hisI. The lack of base substitutions supported the idea that polymerase-defective polA is a minus frameshift- and deletion-type mutator. Contrary to a previous report, we did not observe superior growth of PolA auxotrophs over their prototrophic progenitors (15 auxotrophs tested). We conclude that the polA1 mutation exerts a powerful mutator activity in this specific genetic context.     

Mutation spectra of chemical mutagens determined by Lac+ reversion assay with Escherichia coli WP3101P-WP3106P tester strains

We previously reported the development of mutation-specific Escherichia coli B tester strains WP3101 to WP3106 from strain WP2uvrA. In this study we constructed their pKM101-containing derivatives WP3101P to WP3106P, and further isolated their rfa derivatives WP4101-WP4106 and WP4101P-WP4106P. The six kinds of F' plasmids (lacI-, lacZ-, proAB+), each of which carries a different lacZ allele, contained in the above strains were originally derived from E. coli K-12 strains CC101-CC106. All the tester strains show Lac- and Trp- phenotype. Assays for transitions and transversions are based upon Lac+ reversion of a specific mutation located within the lacZ gene on an F' plasmid. The trpE65(ochre) allele in the same strains enables them to be used for Trp+ reversion assays as well. In the present paper, we evaluated the sensitivity, specificity, and usefulness of the newly developed tester strains. Strains WP3101P-WP3106P were highly sensitive to determine mutational profile of heterocyclic amines with S9 mix-mediated metabolic activation and most of the oxidative mutagens and free radical generators tested. Every type of base-pair substitutions induced by 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) or 5-diazouracil were detected in strains WP3101P-WP3106P, while A:T-->C:G and G:C-->A:T mutations induced by MeIQ, and A:T-->C:G, G:C-->A:T, and G:C-->C:G by 5-diazouracil were not detected in pKM101-free tester strains. In pKM101-carrying strains, cumene hydroperoxide induced all types of base substitutions, while formaldehyde preferentially induced G:C-->T:A transversions. Phenazine methosulfate induced predominantly G:C-->A:T transitions and G:C-->T:A transversions, while H2O2 induced predominantly G:C-->T:A and A:T-->T:A transversions. Introduction of the rfa mutation considerably enhanced sensitivity to bulky mutagens such as polycyclic aromatic compounds. All six possible base substitutions induced by 9, 10-dimethyl-1,2-benzanthracene (DMBA) were detected in tester strains WP4101P-WP4106P. In conclusion, our tester strains WP3101P-WP3106P and WP4101P-WP4106P permitted rapid and simple detection of specific mutations induced by variety of mutagens.     

Sequence specificity of Hprt lymphocyte mutation in rats fed the hepatocarcinogen 2-acetylaminofluorene

Rats fed the hepatocarcinogen 2-acetylaminofluorene (2-AAF) have a low, but significantly increased, frequency of lymphocyte Hprt mutants. In this study, mutants from 2-AAF-fed and control F344 rats were examined for mutations in the Hprt gene in order to determine if the 2-AAF treatment resulted in an agent-specific mutation profile. The most common mutation from 2-AAF-treated rats was G:C-->T:A transversion (32% of all mutations) followed by 1-basepair (bp) deletion (19%); there were very few (5%) G:C-->A:T transitions. Among mutations from control rats, G:C-->A:T transition was the most common (43%), and there were very few G:C-->T:A transversions (5%) and no 1-bp deletions. The profile of mutations from 2-AAF-fed rats was significantly different from control rats (P = 0.003) and was consistent with the types of mutations produced by 2-AAF in vitro. The results of this study indicate that even weak mutational responses in the lymphocyte Hprt assay are capable of producing mutation profiles that reflect the DNA damage inducing them.     

Miscoding and misincorporation of 8-oxo-guanine during leading and lagging strand synthesis in Escherichia coli

We examined whether strand identity with respect to DNA replication influences strand bias for 8-oxo-7,8-dihydroguanine (8-oxoG) mutagenesis. The specificity of 8-oxoG mutagenesis was determined in a mutM mutY or a mutT strain carrying the supF gene on one of two vectors that differed only in the orientation of supF with respect to a unique origin of replication. Most of the supF mutations in the mutM mutY strain were base substitutions (67%), predominantly G:C-->T:A transversions (> 64%), while the majority in the mutT strain were base substitutions (> 92%), predominantly A:T-->C:G transversions (> 91%). The distributions of frequently mutated sites of G:C-->T:A and A:T-->C:G transversions in the supF gene in the mutM mutY and mutT strains, respectively, did not differ markedly between the two vectors. These results suggest that gene orientation is not an important determinant of the strand bias of 8-oxoG mutagenesis.