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.     

A:T --> G:C base pair substitutions occur at a higher rate than other substitution events in Pms2 deficient mouse cells

The mismatch repair pathway involves multiple proteins that are required to correct DNA polymerase generated mismatches before they become mutations. It has been shown recently, that the predominant base-pair substitution events leading to loss of endogenous Aprt activity in Pms2 null mouse cells are A:T --> G:C mutations (Oncogene 21 (2002) 1768, Oncogene 21 (2002) 2840). To determine if this observation could be explained by an increased rate of A:T --> G:C mutations relative to other base-pair substitutions, we developed a reversion assay to examine G:C --> A:T, C:G --> A:T, and A:T --> G:C mutations within mouse Aprt in a Pms2 null mouse kidney cell line. The results demonstrated a 6-50-fold increase in the rate of the A:T --> G:C mutations relative to the other base-pair substitutions. Additional work demonstrated that growth of the Pms2 null cells in antioxidant containing medium reduced the rate of the A:T --> G:C mutations. The results are discussed with regards to the role of mismatch repair proteins in preventing base-pair substitutions, including those induced by oxidative stress.     

Mutation spectra induced by 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide in the supF gene of human XP-A fibroblasts

1-Nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide are oxidative metabolites that are responsible for the mutagenicity of 1-nitropyrene. In this study, the mutation spectra induced by oxidative metabolites in human cells were determined using a shuttle vector assay. The mutation frequencies induced by 1-nitropyrene 9,10-oxide were 2-3 times higher than those induced by 1-nitropyrene 4,5-oxide. The base substitutions induced by 1-nitropyrene 4,5-oxide were G --> A transitions, G --> C transversions, and G --> T transversions. In the case of 1-nitropyrene 9,10-oxide, G --> A transitions, G --> T transversions, A --> G transitions and G --> C transversions were observed. Most base substitution mutations induced by oxidative metabolites occurred at the guanine sites in the supF gene. These sequence-specific hot spots were commonly identified as 5'-GA sequences for both metabolites. On the other hand, the sequence-specific hot spots at the adenine sites were identified as 5'-CAC sequences for 1-nitropyrene 9,10-oxide. These results suggest that the oxidative metabolites of 1-nitropyrene induce sequence-specific DNA mutations at the guanine and adenine sites at high frequency.     

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.     

Estimation of DNA Sequence Context-dependent Mutation Rates Using Primate Genomic Sequences

It is understood that DNA and amino acid substitution rates are highly sequence context-dependent, e.g., C --> T substitutions in vertebrates may occur much more frequently at CpG sites and that cysteine substitution rates may depend on support of the context for participation in a disulfide bond. Furthermore, many applications rely on quantitative models of nucleotide or amino acid substitution, including phylogenetic inference and identification of amino acid sequence positions involved in functional specificity. We describe quantification of the context dependence of nucleotide substitution rates using baboon, chimpanzee, and human genomic sequence data generated by the NISC Comparative Sequencing Program. Relative mutation rates are reported for the 96 classes of mutations of the form 5' alphabetagamma 3' --> 5' alphadeltagamma 3', where alpha, beta, gamma, and delta are nucleotides and beta not equal delta, based on maximum likelihood calculations. Our results confirm that C --> T substitutions are enhanced at CpG sites compared with other transitions, relatively independent of the identity of the preceding nucleotide. While, as expected, transitions generally occur more frequently than transversions, we find that the most frequent transversions involve the C at CpG sites (CpG transversions) and that their rate is comparable to the rate of transitions at non-CpG sites. A four-class model of the rates of context-dependent evolution of primate DNA sequences, CpG transitions > non-CpG transitions approximately CpG transversions > non-CpG transversions, captures qualitative features of the mutation spectrum. We find that despite qualitative similarity of mutation rates among different genomic regions, there are statistically significant differences.     

Escherichia coli strains (ndk) lacking nucleoside diphosphate kinase are powerful mutators for base substitutions and frameshifts in mismatch-repair-deficient strains

Nucleoside diphosphate (NDP) kinase is one of the enzymes that maintains triphosphate pools. Escherichia coli strains (ndk) lacking this enzyme have been shown to be modest base substitution mutators, and two members of the human family of NDP kinases act as tumor suppressors. We show here that in E. coli strains lacking NDP kinase high levels of mispairs are generated, but most of these are corrected by the mismatch-repair system. Double mutants that are ndk mutS, lacking both the NDP kinase and mismatch repair, have levels of base substitutions 15-fold higher and levels of certain frameshifts up to 10-fold higher than those of the respective mutations in mutS strains that are NDP kinase proficient. A sequence analysis of the specificity of base substitution mutations generated in ndk and ndk mutS backgrounds as well as other experiments suggests that NDP kinase deficiency stimulates polymerase errors that lead to A:T --> G:C transitions and that the editing capacity of cells may be affected, leading to additional uncorrected mispairs and to A:T --> T:A transversions.     

Mutagenicity of the cytidine analog zebularine in Escherichia coli

We have examined the mutagenic properties of zebularine, a cytidine analog lacking the amino group at C-4 that has potential use in chemotherapy. Because the hydrate is a strong inhibitor of cytidine deaminase, its use can enhance the potency of other cytosine based compounds such as 5-azacytidine (5AzaC) and cytosine arabinoside (ara-C) that are inactivated by cytidine deaminase. Using the newly developed rpoB/Rifr system in Escherichia coli, we examined base substitution mutations caused by zebularine in the chromosomal rpoB gene. Zebularine is a potent mutagen that causes mainly G : C --> A : T transitions and favors certain hotspots. Mutations are not specific to the rpoB gene, since there is also a strong induction of mutations in the thyA gene. In the absence of mismatch repair, zebularine induces both base substitutions and frame shifts at rates well above those seen in wild-type strains treated with zebularine or in mismatch repair deficient strains without treatment. The nature of these induced mutations indicates that zebularine is stimulating the induction of increased replication errors, in addition to the targeted G : C --> A : T mutations, and that these errors are normally repaired by the mismatch repair system.     

The complementary neighborhood patterns and methylation-to-mutation likelihood structures of 15,110 single-nucleotide polymorphisms in the bovine genome

Bayesian analysis was performed to examine the single-nucleotide polymorphism (SNPs) neighborhood patterns in cattle using 15,110 SNPs, each with a flanking sequence of 500 bp. Our analysis confirmed three well-known features reported in plants and/or other animals: (1) the transition is the most abundant type of SNPs, accounting for 69.8% in cattle; (2) the transversion occurs most frequently (38.56%) in cattle when the A + T content equals two at their immediate adjacent sites; and (3) C <--> T and A <--> G transitions have reverse complementary neighborhood patterns and so do A <--> C and G <--> T transversions. Our study also revealed several novel SNP neighborhood patterns that have not been reported previously. First, cattle and humans share an overall SNP pattern, indicating a common mutation system in mammals. Second, unlike C <--> T/A <--> G and A <--> C/G <--> T, the true neighborhood patterns for A <--> T and C <--> G might remain mysterious because the sense and antisense sequences flanking these mutations are not actually recognizable. Third, among the reclassified four types of SNPs, the neighborhood ratio between A + T and G + C was quite different. The ratio was lowest for C <--> G, but increased for C <--> T/A <--> G, further for A <--> C/G <--> T, and the most for A <--> T. Fourth, when two immediate adjacent sites provide structures for CpG, it significantly increased transitions compared to the structures without the CpG. Finally, unequal occurrence between A <--> G and C <--> T in five paired neighboring structures indicates that the methylation-induced deamination reactions were responsible for approximately 20% of total transitions. In addition, conversion can occur at both CpG sites and non-CpG sites. Our study provides new insights into understanding molecular mechanisms of mutations and genome evolution.     

Mutational specificity of the base analogue, 2-aminopurine,in Escheichica coli

2-Aminopurine (2-AP) is a base analogue of adenine which mispairs with cytosine and causes base-pair substitutions of the transition type. By analyzing the reversion patterns of defined trpA alleles in Escheriachia coli we confirm that 2-AP cuases both A:T → G:C and G:C → A:T transitions whith the former induced more frequently than the latter. We also find that 2-AP enhances transversion at 3 sites and frameshift mutations at 1 other site. It is unlikely that 2-AP can cause transversions and frameshifts solely by a mispairing mechanism. However, 2-AP-induced transversion and frameshift mutagenesis was not abolished by the presence of an inactive recA allele, indicating this mutagenic activity is not dependent upon recA-directed misrepair.     

Changes in DNA Base Sequence Induced by Gamma-Ray Mutagenesis of Lambda Phage and Prophage

Mutations in the cI (repressor) gene were induced by gamma-ray irradiation of lambda phage and of prophage, and 121 mutations were sequenced. Two-thirds of the mutations in irradiated phage assayed in recA host cells (no induction of the SOS response) were G:C to A:T transitions; it is hypothesized that these may arise during DNA replication from adenine mispairing with a cytosine product deaminated by irradiation. For irradiated phage assayed in host cells in which the SOS response had been induced, 85% of the mutations were base substitutions, and in 40 of the 41 base changes, a preexisting base pair had been replaced by an A:T pair; these might come from damaged bases acting as AP (apurinic or apyrimidinic) sites. The remaining mutations were 1 and 2 base deletions. In irradiated prophage, base change mutations involved the substitution of both A:T and of G:C pairs for the preexisting pairs; the substitution of G:C pairs shows that some base substitution mechanism acts on the cell genome but not on the phage. In the irradiated prophage, frameshifts and a significant number of gross rearrangements were also found.     

Characterization of TCHQ-induced genotoxicity and mutagenesis using the pSP189 shuttle vector in mammalian cells

Tetrachlorohydroquinone (TCHQ) is a major toxic metabolite of the widely used wood preservative, pentachlorophenol (PCP), and it has also been implicated in PCP genotoxicity. However, the underlying mechanisms of genotoxicity and mutagenesis induced by TCHQ remain unclear. In this study, we examined the genotoxicity of TCHQ by using comet assays to detect DNA breakage and formation of TCHQ-DNA adducts. Then, we further verified the levels of mutagenesis by using the pSP189 shuttle vector in A549 human lung carcinoma cells. We demonstrated that TCHQ causes significant genotoxicity by inducing DNA breakage and forming DNA adducts. Additionally, DNA sequence analysis of the TCHQ-induced mutations revealed that 85.36% were single base substitutions, 9.76% were single base insertions, and 4.88% were large fragment deletions. More than 80% of the base substitutions occurred at G:C base pairs, and the mutations were G:C to C:G, G:C to T:A or G:C to A:T transversions and transitions. The most common types of mutations in A549 cells were G:C to A:T (37.14%) and A:T to C:G transitions (14.29%) and G:C to C:G (34.29%) and G:C to T:A (11.43%) transversions. We identified hotspots at nucleotides 129, 141, and 155 in the supF gene of plasmid pSP189. These mutation hotspots accounted for 63% of all single base substitutions. We conclude that TCHQ induces sequence-specific DNA mutations at high frequencies. Therefore, the safety of using this product would be carefully examined.     

Methylglyoxal induces G:C to C:G and G:C to T:A transversions in the supF gene on a shuttle vector plasmid replicated in mammalian cells

We previously reported that the majority of base-pair substitutions induced by an endogenous mutagen, methylglyoxal, were G:C-->T:A transversions and G:C-->A:T transitions in wild-type and nucleotide excision repair (NER)-deficient (uvrA or uvrC) Escherichia coli strains. To investigate the mutation spectrum of methylglyoxal in mammalian cells and to compare the spectrum with those detected in other experimental systems, we analyzed mutations in a bacterial suppressor tRNA (supF) gene in the shuttle vector plasmid pMY189. We treated pMY189 with methylglyoxal and immediately transfected it into simian COS-7 cells. The cytotoxicity and the mutation frequency (MF) increased according to the dose of methylglyoxal. In the mutants induced by methylglyoxal, multi-base deletions were predominant (50%), followed by base-pair substitutions (35%), in which 89% of the substitutions occurred at G:C sites. Among them, G:C-->C:G and G:C-->T:A transversions were predominant. The overall distribution of methylglyoxal-induced mutations detected in the supF gene was different from that for the spontaneous mutations. These results suggest that methylglyoxal may take part in causing G:C-->C:G and G:C-->T:A transversions in vivo.     

Patterns of nucleotide substitution in pseudogenes and functional genes

Summary The pattern of point mutations is inferred from nucleotide substitutions in pseudogenes. The pattern obtained suggests that transition mutations occur somewhat more frequently than transversion mutations and that mutations result more often in A or T than in G or C. Our results are discussed with respect to the predictions from Topal and Fresco's model for the molecular basis of point (substitution) mutations (Nature 263:285–289, 1976). The pattern of nucleotide substitution at the first and second positions of codons in functional genes is quite similar to that in pseudogenes, but the relative frequency of the transition CT in the sense strand is drastically reduced and those of the transversions CG and GC are doubled. The differences between the two patterns can be explained by the observation that in the protein evolution amino acid substitutions occur mainly between amino acids with similar biochemical properties (Grantham, Science 185:862–864, 1974). Our results for the patterns of nucleotide substitutions in pseudogenes and in functional genes lead to the prediction that both the coding and non-coding regions of protein coding genes should have high frequencies of A and T. Available data show that the non-coding regions are indeed high in A and T but the coding regions are low in T, though high in A.     

Novel mutational spectrum induced by N-methyl-N'-nitro-N-nitrosoguanidine in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase gene in diploid human fibroblasts

The kinds and locations of mutations in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase (hprt) gene of 75 independent mutants, derived from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated normal human fibroblasts, were characterized by direct sequencing of mRNA-polymerase chain reaction (mRNA-PCR)-amplified cDNA. Treatment of human cells with low (6 or 8 microM) or high (10 or 12 microM) doses of MNNG resulted in 35-fold or 150-fold average increases in mutation frequency, respectively. A high frequency of mutants lacking a complete exon was observed in both groups. Further characterization of half of these mutants by DNA-PCR amplification of intron-exon boundaries showed that they contained base substitutions. The kinds of base substitutions differed distinctly between these two groups. In the low dose group, a broad mutational spectrum was observed: ten out of the 31 base substitutions were A.T to G.C transitions, six contained G.C to A.T transitions, and the other 15 exhibited transversions. In contrast, the majority (84%) of base substitutions among the high dose group were G.C to A.T transitions; the others (16%) were transversions. All of the 32 G.C to A.T transitions were located on the non-transcribed strand, assuming that the causative premutational lesion was O6-methylguanine. These results indicate preferential repair of lesions located on the transcribed strand. In addition, G.C to A.T and A.T to G.C transitions preferentially occurred at positions with guanine and thymine at the adjacent 5' position, respectively.     

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.     

Characterization of mutational specificity within the lacI gene for a mutD5 mutator strain of Escherichia coli defective in 3''----5'' exonuclease (proofreading) activity.

The mutD (dnaQ) gene of Escherichia coli codes for the epsilon subunit of the DNA polymerase III holoenzyme which is involved in 3'----5' exonuclease proofreading activity. We determined the mutational specificity of the mutator allele, mutD5, in the lacI gene of E. coli. The mutD5 mutation preferentially produces single base substitutions as judged from the enhanced fraction of lacI nonsense mutations and the spectrum of sequenced dominant lacI (lacId) and constitutive lacO (lacOc) mutations which were predominantly (69/71) single nucleotide substitutions. The distribution of amber lacI and sequenced lacId mutations revealed that transitions occur more frequently than transversions. A . T----G . C and G . C----A . T transitions were equally frequent and, with one major exception, evenly distributed among numerous sites. Among the transversions, A . T----T . A events were the most common, A . T----C . G substitutions were rare, and G . C----C . G changes were not detected. Transversions were unequally distributed among a limited number of sites with obvious hotspots. All 11 sequenced transversions had a consensus neighboring sequence of 5'-C-C-(mutated G or A)-C-3'. Although no large deletions or complex mutational events were recovered, sequencing revealed that mutD5 induced single nucleotide deletions within consecutive G X C sequences. An extraordinary A . T----G . C transition hotspot occurred at nucleotide position +6 in the lac operator region; the mutD5 mutation frequency of this single base pair was calculated to be 1.2 X 10(-3).     

Mitochondrial DNA mutations in the parotid gland of cigarette smokers and non-smokers

It has previously been demonstrated that mitochondrial DNA (mtDNA) mutations accumulate in the lung and increase in frequency with age. It has also been shown that the level of mtDNA mutations including deletions and base substitutions are elevated in lung tissue of smokers relative to non-smokers. We have previously shown that the 'common' 4977 bp mtDNA deletion is present in the parotid (salivary) gland of smokers and non-smokers and that there is a significant increase in the level of this deletion in Warthins tumour, an oncocytoma of the parotid gland. In this study we used semi-quantitative PCR to confirm the presence of 4977 bp mtDNA deletion in the parotid gland of non-smokers and smokers. Importantly, we show that the deletion accumulates with age regardless of smoking status and that there was no significant difference in the level of the 4977 bp deletion in parotid tissue of smokers and non-smokers. Using strand conformational polymorphism (SSCP) and direct sequencing we also found 5/23 smokers had parotid tissue specific base substitutions: either an A/T to G/C transition at A4767 or a G/C to A/T transition at G4853. These results are evidence of age related increase in the 4977 bp deletion and a higher level of mutations, probably due to oxidative damage, in the parotid gland of smokers.