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.     

Analysis of spontaneous base substitutions generated in mutator strains of Bacillus subtilis

In the current studies, we investigated base substitutions in the Bacillus subtilis mutT, mutM, and mutY DNA error-prevention system. In the wild type strain, spontaneous mutations were mainly transitions, either G:C --> A:T or A:T --> G:C. Although both transitions and transversions were observed in mutY and mutM mutants, mutM/mutY double mutants contain strictly G:C --> T:A transversions. In the mutT strain, A:T --> C:G transversion was not observed, and over-expression of the B. subtilis mutT gene had no effect on the mutation rate in the Escherichia coli mutT strain. Using 8-oxo-dGTP-induced mutagenesis, transitions especially A:T --> G:C were predominant in the wild type and mutY strains. In contrary, transversion was high on mutY and double mutant (mutM mutY). Finally, the opuBC and yitG genes were identified from the B. subtilis chromosome as mutator genes that prevented the transition base substitutions.     

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.     

Escherichia coli MutY protein has a guanine-DNA glycosylase that acts on 7,8-dihydro-8-oxoguanine:guanine mispair to prevent spontaneous G:C-->C:G transversions.

Low rates of spontaneous G:C-->C:G transversions would be achieved not only by the correction of base mismatches during DNA replication but also by the prevention and removal of oxidative base damage in DNA. Escherichia coli must have several pathways to repair such mismatches and DNA modifications. In this study, we attempted to identify mutator loci leading to G:C-->C:G transversions in E.coli. The strain CC103 carrying a specific mutation in lacZ was mutagenized by random miniTn 10 insertion mutagenesis. In this strain, only the G:C-->C:G change can revert the glutamic acid at codon 461, which is essential for sufficient beta-galactosidase activity to allow growth on lactose. Mutator strains were detected as colonies with significantly increased rates of papillae formation on glucose minimal plates containing P-Gal and X-Gal. We screened approximately 40 000 colonies and selected several mutator strains. The strain GC39 showed the highest mutation rate to Lac+. The gene responsible for the mutator phenotypes, mut39 , was mapped at around 67 min on the E.coli chromosome. The sequencing of the miniTn 10 -flanking DNA region revealed that the mut39 was identical to the mutY gene of E.coli. The plasmid carrying the mutY + gene reduced spontaneous G:C-->T:A and G:C-->C:G mutations in both mutY and mut39 strains. Purified MutY protein bound to the oligonucleotides containing 7,8-dihydro-8-oxo-guanine (8-oxoG):G and 8-oxoG:A. Furthermore, we found that the MutY protein had a DNA glycosylase activity which removes unmodified guanine from the 8-oxoG:G mispair. These results demonstrate that the MutY protein prevents the generation of G:C-->C:G transversions by removing guanine from the 8-oxoG:G mispair in E.coli.     

Characterization of the GO system of Pseudomonas aeruginosa

The mutT, mutM, and mutY genes of the GO system of the Pseudomonas aeruginosa PAO1 strain have been characterized by cloning, sequencing, and complementation analysis. The three genes, when cloned in a plasmid, were able to complement the high mutation frequency of the corresponding Escherichia coli deficient strains. Our results demonstrate that the putative mutT, mutM, and mutY gene products from P. aeruginosa are able to perform the expected activity. In addition, the sequence of the P. aeruginosa mutT gene strongly suggested that the product of this gene has a bifunctional activity in P. aeruginosa, being the C-terminal part 40% identical to a consensus sequence of thiamine monophosphate synthases. Our results also demonstrated that the N-terminal part of the protein is necessary and sufficient for the 8-oxodGTP hydrolase activity.     

A Novel Role for Escherichia coli Endonuclease VIII in Prevention of Spontaneous G→T Transversions

In the bacterium Escherichia coli, oxidized pyrimidines are removed by two DNA glycosylases, endonuclease III and endonuclease VIII (endo VIII), encoded by the nth and nei genes, respectively. Double mutants lacking both of these activities exhibit a high spontaneous mutation frequency, and here we show that all of the mutations observed in the double mutants were G:C-->A:T transitions; no thymine mutations were found. These findings are in agreement with the preponderance of C-->T transitions in the oxidative and spontaneous mutational databases. The major oxidized purine lesion in DNA, 7,8-dihydro-8-oxoguanine (8-oxoG), is processed by two DNA glycosylases, formamidopyrimidine DNA glycosylase (Fpg), which removes 8-oxoG opposite C, and MutY DNA glycosylase, which removes misincorporated A opposite 8-oxoG. The high spontaneous mutation frequency previously observed in fpg mutY double mutants was significantly enhanced by the addition of the nei mutation, suggesting an overlap in the substrate specificities between endo VIII and Fpg/MutY. When the mutational specificity was examined, all of the mutations observed were G:C-->T:A transversions, indicating that in the absence of Fpg and MutY, endo VIII serves as a backup activity to remove 8-oxoG. This was confirmed by showing that, indeed, endo VIII can recognize 8-oxoG in vitro.     

Mutagenesis and repair in Bacillus anthracis: the effect of mutators

We have generated mutator strains of Bacillus anthracis Sterne by using directed gene knockouts to investigate the effect of deleting genes involved in mismatch repair, oxidative repair, and maintaining triphosphate pools. The single-knockout strains are deleted for mutS, mutY, mutM, or ndk. We also made double-knockout strains that are mutS ndk or mutY mutM. We have measured the levels of mutations in the rpoB gene that lead to the Rif(r) phenotype and have examined the mutational specificity. In addition, we examined the mutational specificity of two mutagens, 5-azacytidine and N-methyl-N'-nitro-N-nitroso-guanidine. The mutY and mutM single knockouts are weak mutators by themselves, but the combination of mutY mutM results in very high mutation rates, all due to G:C --> T:A transversions. The situation parallels that seen in Escherichia coli. Also, mutS knockouts are strong mutators and even stronger in the presence of a deletion of ndk. The number of sites in rpoB that can result in the Rif(r) phenotype by single-base substitution is more limited than in certain other bacteria, such as E. coli and Deinococcus radiodurans, although the average mutation rate per mutational site is roughly comparable. Hotspots at sites with virtually identical surrounding sequences are organism specific.     

Reduction of GC --> TA transversion mutation by overexpression of MutS in Escherichia coli K-12

Overexpression of the MutS repair protein significantly decreased the rate of lacZ GC --> TA transversion mutation in stationary-phase and exponentially growing bacteria and in mutY and mutM mutants, which accumulate mismatches between 8-oxoguanine (8-oxoG) and adenine residues in DNA. Conversely, GC --> TA transversion increased in mutL or mutS mutants in stationary phase. In contrast, overexpression of MutS did not appreciably reduce lacZ AT --> CG transversion mutation in a mutT mutant. These results suggest that MutS-dependent repair can correct 8-oxoG:A mismatches in Escherichia coli cells but may not be able to compete with mutation fixation by MutY in mutT mutants.     

Mutagenesis and carcinogenesis caused by the oxidation of nucleic acids

Genomes and their precursor nucleotides are highly exposed to reactive oxygen species, which are generated both as byproducts of oxygen respiration or molecular executors in the host defense, and by environmental exposure to ionizing radiation and chemicals. To counteract such oxidative damage in nucleic acids, mammalian cells are equipped with three distinct enzymes. MTH1 protein hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-2'-deoxyguanosine triphosphate and 2-hydroxy-2'-deoxyadenosine triphosphate (2-OH-dATP), to the corresponding monophosphates. We observed increased susceptibility to spontaneous carcinogenesis in MTH1-null mice, which exhibit an increased occurrence of A:T-->C:G and G:C-->T:A transversion mutations. 8-Oxoguanine (8-oxoG) DNA glycosylase, encoded by the OGG1 gene, and adenine DNA glycosylase, encoded by the MUTYH gene, are responsible for the suppression of G:C to T:A transversions caused by the accumulation of 8-oxoG in the genome. Deficiency of these enzymes leads to increased tumorigenesis in the lung and intestinal tract in mice, respectively. MUTYH deficiency may also increase G:C to T:A transversions through the misincorporation of 2-OH-dATP, especially in the intestinal tract, since MUTYH can excise 2-hydroxyadenine opposite guanine in genomic DNA and the repair activity is selectively impaired by a mutation found in patients with autosomal recessive colorectal adenomatous polyposis.     

Delayed transfection of DNA after riboflavin mediated photosensitization increases G:C to C:G transversions of supF gene in Escherichia coli mutY strain.

We have previously reported that the majority of base substitution mutations of the Escherichia coli supF gene induced by riboflavin mediated photosensitization were G:C to C:G changes, in addition to G:C to T:A changes which were probably caused by 8-hydroxyguanine (oh(8)Gua), in wild type and mutM mutator mutant strains. This implies that lesions other than oh(8)Gua are produced by riboflavin-photosensitization. G:C to C:G base substitutions have been found in the mutations induced by ionizing radiation and reactive oxygen species, as well as spontaneous mutation. To characterize the G:C to C:G mutation, riboflavin- photosensitized plasmid DNA carrying the supF gene was left at room temperature for 5 h in the dark before transfection. The delayed transfection gave a mutational spectrum different from that for immediate transfection. G:C to C:G transversions significantly increased in mutY mutator strain, in which the transversion was not detected in the immediate transfection. Lesions causing G:C to C:G changes increased during 5-h holding after photosensitization and MutY protein presumably takes part in this type of base change mutation.     

Escherichia coli mutT mutator effect during in vitro DNA synthesis. Enhanced A.G replicational errors

The mechanism of the Escherichia coli mutT mutator effect was investigated using single-stranded phage as a mutational target. In vivo experiments showed that two M13mp2 lacZ alpha nonsense mutants reverted at a higher rate on a mutT1 host than on the wild-type host. The specificity of this mutator effect was identical to that observed for E. coli genes: A.T----C.G transversions. The mutT effect was subsequently demonstrated in vitro during DNA replication of M13mp2 DNA in cell-free extracts of E. coli. Replication (the single-stranded----replicative form conversion) in mutT1 extracts proceeded with a higher error rate than in wild-type extracts, and DNA sequence analysis of the in vitro revertants revealed the specific induction of A.T----C.G transversions. On the basis of the template specificity of the mutT effect in vitro, we conclude that the mutT effect involves the aberrant processing of A.G rather than T.C mispairs.     

The GO system prevents ROS-induced mutagenesis and killing in Pseudomonas aeruginosa

Inactivation of the Pseudomonas aeruginosa mutM, mutY , or mutT gene conferred a 2.4-, 17.2-, or 38.1-fold increase in spontaneous mutation frequency, respectively. Importantly, the mutY and mutT strains each displayed a robust H₂O₂-induced mutation frequency. In addition, the mutM, mutY , and mutT mutations severely sensitized P. aeruginosa to killing by H₂O₂, suggesting that these gene products act to repair one or more cytotoxic lesions in P. aeruginosa . Nucleotide sequence analysis of a fragment of the rpoB gene from rifampicin resistant mutM -, mutY -, and, mutT -deficient strains was consistent with this conclusion. These findings are discussed in terms of possible roles for mutM, mutY , and mutT in contributing to survival and mutagenesis of P. aeruginosa colonizing the airways of cystic fibrosis patients.     

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.     

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.     

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.     

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.     

Opposite base-dependent reactions of a human base excision repair enzyme on DNA containing 7,8-dihydro-8-oxoguanine and abasic sites.

The guanine modification 7,8-dihydro-8-oxoguanine (8-oxoG) is a potent premutagenic lesion formed spontaneously at high frequencies in the genomes of aerobic organisms. We have characterized a human DNA repair glycosylase for 8-oxoG removal, hOGH1 (human yeast OGG1 homologue), by molecular cloning and functional analysis. Expression of the human cDNA in a repair deficient mutator strain of Escherichia coli (fpg mutY) suppressed the spontaneous mutation frequency to almost normal levels. The hOGH1 enzyme was localized to the nucleus in cells transfected by constructs of hOGH1 fused to green fluorescent protein. Enzyme purification yielded a protein of 38 kDa removing both formamidopyrimidines and 8-oxoG from DNA. The enzymatic activities of hOGH1 was analysed on DNA containing single residues of 8-oxoG or abasic sites opposite each of the four normal bases in DNA. Excision of 8-oxoG opposite C was the most efficient and was followed by strand cleavage via beta-elimination. However, significant removal of 8-oxoG from mispairs (8-oxoG: T >G >A) was also demonstrated, but essentially without an associated strand cleavage reaction. Assays with abasic site DNA showed that strand cleavage was indeed dependent on the presence of C in the opposite strand, irrespective of the prior removal of an 8-oxoG residue. It thus appears that strand incisions are made only if repair completion results in correct base insertion, whereas excision from mispairs preserves strand continuity and hence allows for error-free correction by a postreplicational repair mechanism.     

mutM, a second mutator locus in Escherichia coli that generates G.C----T.A transversions.

We used strains carrying specific lacZ alleles to identify a new mutator locus in Escherichia coli which generates only G.C----T.A transversions among base substitutions. The locus, mutM, mapped near the cysE locus, which is at 81 min on the genetic map.     

Bacterial DNA repair genes and their eukaryotic homologues: 2. Role of bacterial mutator gene homologues in human disease. Overview of nucleotide pool sanitization and mismatch repair systems

Since the discovery of the first E. coli mutator gene, mutT, most of the mutations inducing elevated spontaneous mutation rates could be clearly attributed to defects in DNA repair. MutT turned out to be a pyrophosphohydrolase hydrolyzing 8-oxodGTP, thus preventing its incorporation into DNA and suppresing the occurrence of spontaneous AT-->CG transversions. Most of the bacterial mutator genes appeared to be evolutionarily conserved, and scientists were continuously searching for contribution of DNA repair deficiency in human diseases, especially carcinogenesis. Yet a human MutT homologue--hMTH1 protein--was found to be overexpressed rather than inactivated in many human diseases, including cancer. The interest in DNA repair contribution to human diseases exploded with the observation that germline mutations in mismatch repair (MMR) genes predispose to hereditary non-polyposis colorectal cancer (HNPCC). Despite our continuously growing knowledge about DNA repair we still do not fully understand how the mutator phenotype contributes to specific forms of human diseases.