Hydrogen peroxide induces G:C to TA and G:C to C:G transversions in the supF gene of Escherichia coli

A vector plasmid, pZ189, carrying an Escherichia coli supF gene as a target for mutations, was treated with a combination of hydrogen peroxide and Fe³⁺/EDTA complex and propagated in E. coli host cells that had been induced for SOS functions by ultraviolet irradiation. The mutations frequency increased by up to 30-fold over spontaneous background levels with increasing concentrations of hydrogen peroxide. The increase in mutation frequency correlated with an increase in the formation of 8-hydroxydeoxyguanosine in the pZ189 DNA. Sequence analysis of 82 independent supF mutant plasmids revealed that 70 mutants contained base substitutions, with 63 of the 70 involving a G:C base pair, and with G:C→C:G (28 cases) and G:C→T:A (26 cases) transversions predominating. Investigation of the influence of the local DNA sequence on the transversions revealed that the guanine at the center of the triplet 5′-PuGA-3′ was five times more likely to mutate after treatment with hydrogen peroxide than that at the center of 5′PyGN3′. G:C→T:A transversions presumably resulted from mispairing of an altered G (probably 8-hydroxydeoxyguanosine) with deoxyadenosine. The origin of the G:C→C:G transversions may be an as yet unidentified lesion generated by hydrogen peroxide. Mutagenic hotspots for base substitutions were found at positions 133, 160 and 168. Mutation spectra and the positions of mutagenic hotspots, when compared with a previously determined spontaneous mutagenesis spectrum, also provide information on the mechanism of spontaneous mutagenesis.     

Repair of Single Base-Pair Transversion Mismatches of Escherichia Coli in Vitro: Correction of Certain a/G Mismatches Is Independent of Dam Methylation and Host Muthls Gene Functions

Six different base-pair transversion mismatches are repaired with different efficiencies in an in vitro mismatch repair system. In particular, the T/T and C/C mismatches appear to be less efficiently repaired than the A/A and G/G mismatches. Four A/G and four C/T mismatches at different positions are repaired to different extents. One of the A/G mismatches is repaired equally efficiently when DNA heteroduplexes are fully methylated or hemi-methylated at the d(GATC) sequences. This type of mismatch repair appears to be unidirectional with A to C conversion by acting at A/G mispairs to restore the C/G pairs. This methylation-independent correction is not controlled by the mutH, mutL, mutS, uvrE, uvrB, phr, recA, recF, and recJ gene products. The independence of the transversion mismatch repair of these genes and methylation distinguishes this from the known mismatch repair pathways.     

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.     

Hydrogen peroxide induces G:C to TA and G:C to C:G transversions in the supF gene of Escherichia coli

A vector plasmid, pZ189, carrying an Escherichia coli supF gene as a target for mutations, was treated with a combination of hydrogen peroxide and Fe³⁺/EDTA complex and propagated in E. coli host cells that had been induced for SOS functions by ultraviolet irradiation. The mutations frequency increased by up to 30-fold over spontaneous background levels with increasing concentrations of hydrogen peroxide. The increase in mutation frequency correlated with an increase in the formation of 8-hydroxydeoxyguanosine in the pZ189 DNA. Sequence analysis of 82 independent supF mutant plasmids revealed that 70 mutants contained base substitutions, with 63 of the 70 involving a G:C base pair, and with G:CC:G (28 cases) and G:CT:A (26 cases) transversions predominating. Investigation of the influence of the local DNA sequence on the transversions revealed that the guanine at the center of the triplet 5-PuGA-3 was five times more likely to mutate after treatment with hydrogen peroxide than that at the center of 5PyGN3. G:CT:A transversions presumably resulted from mispairing of an altered G (probably 8-hydroxydeoxyguanosine) with deoxyadenosine. The origin of the G:CC:G transversions may be an as yet unidentified lesion generated by hydrogen peroxide. Mutagenic hotspots for base substitutions were found at positions 133, 160 and 168. Mutation spectra and the positions of mutagenic hotspots, when compared with a previously determined spontaneous mutagenesis spectrum, also provide information on the mechanism of spontaneous mutagenesis.     

Statistical evidence for a biochemical pathway of natural, sequence-targeted G/C to C/G transversion mutagenesis in Haemophilus influenzae Rd.

Markov chain analysis of the Haemophilus influenzae Rd genome reveals striking under-representation of three palindromic tetranucleotide strings (CCGG, GGCC and CATG), accompanied by over-representation of six tetranucleotide strings that are derived from the former by exchanging strand location of the two residues making up a G/C nucleotide pair at the terminal palindrome position. Constraints are outlined for a molecular model able to explain the phenomenon as the result of sequence-targeted, enzyme-driven G/C to C/G transversion mutagenesis. Possible participation in the process by components of known DNA mismatch repair or restriction/modification systems (in particular, cytosine methylation) is discussed. The effect widens the spectrum of enzyme-driven, specific mutagenesis beyond the formerly described C/G to T/A transition (VSP repair of Escherichia coli). Potential evolutionary benefits of enzymatic pathways of specific mutagenesis can be envisioned.     

A DNA repair process in Escherichia coli corrects U:G and T:G mismatches to C:G at sites of cytosine methylation

Escherichia coli contains a base mismatch correction system called VSP repair that is known to correct T:G mismatches to C:G when they occur in certain sequence contexts. The preferred sequence context for this process is the site for methylation by the E. coli DNA cytosine methylase (Dcm). For this reason, VSP repair is thought to counteract potential mutagenic effects of deamination of 5-methylcytosine to thymine. We have developed a genetic reversion assay that quantitates the frequency of C to T mutations at Dcm sites and the removal of such mutations by DNA repair processes. Using this assay, we have studied the repair of U: G mismatches in DNA to C: G and have found that VSP repair is capable of correcting these mismatches. Although VSP repair substantially affects the reversion frequency, it may not be as efficient at correcting U: G mismatches as the uracil DNA glycosylase-mediated repair process.     

Repair of a Mismatch Is Influenced by the Base Composition of the Surrounding Nucleotide Sequence

Heteroduplexes with single base pair mismatches of known sequence were prepared by annealing separated strands of bacteriophage lambda DNA and used to transfect Escherichia coli. A series of transition (G:T and A:C) and transversion (G:A and C:T) mismatches located throughout most of the bacteriophage lambda cI gene has been examined. The results suggest that the transition mismatches are generally better repaired than the transversion mismatches and that, at least for the transversion mismatches studied, repair efficiency increases with increasing G:C content in the neighboring nucleotide sequence. This specificity of the E. coli mismatch repair system can account, in part, for the similar frequencies of base substitution mutations throughout the E. coli genome.     

Niemann-Pick type C disease: mutations of NPC1 gene and evidence of abnormal expression of some mutant alleles in fibroblasts

We analyzed Niemann-Pick type C disease 1 (NPC1) gene in 12 patients with Niemann-Pick type C disease by sequencing both cDNA obtained from fibroblasts and genomic DNA. All the patients were compound heterozygotes. We found 15 mutations, eight of which previously unreported. The comparison of cDNA and genomic DNA revealed discrepancies in some subjects. In two unrelated patients carrying the same mutations (P474L and nt 2972del2) only one mutant allele (P474L), was expressed in fibroblasts. The mRNA corresponding to the other allele was not detected even in cells incubated with cycloheximide. The promoter variants (-1026T/G and -1186T/C or -238 C/G), found to be in linkage with 2972del2 allele do not explain the lack of expression of this allele, as they were also found in control subjects. In another patient, (N1156S/Q922X) the N1156S allele was expressed in fibroblasts while the expression of the other allele was hardly detectable. In a fourth patient cDNA analysis revealed a point mutation in exon 20 (P1007A) and a 56 nt deletion in exon 22 leading to a frameshift and a premature stop codon. The first mutation was confirmed in genomic DNA; the second turned out to be a T-->G transversion in exon 22, predicted to cause a missense mutation (V1141G). In fact, this transversion generates a donor splice site in exon 22, which causes an abnormal pre-mRNA splicing leading to a partial deletion of this exon. In some NPC patients, therefore, the comparison between cDNA and genomic DNA may reveal an unexpected expression of some mutant alleles of NPC1 gene.     

General nearest neighbor preferences in G/C oligomers interrupted by A/T: correlation with DNA structure

The frequencies of occurrence of the 5' and 3' nearest neighbor doublets of oligonucleotides containing (G/C) and (A/T) blocks show strong trends. Specifically, the following trends are observed. Given a (G/C)n (A/T)m oligomer (where G/C)n indicates a sequence of length n composed solely of Gs and/or Cs and (A/T)m is a sequence of length m composed solely of As and/or Ts, and n = 3,2,1; m = 1,2,3) and a (G/mC)2 doublet, (G/C)n (A/T)m (G/C)2 greater than (G/C)n + 2 (A/T)m. That is the (G/C)2 doublet is preferentially located 3' of the oligomer, enclosing the (A/T)m stretch. The trends are strongest for n = 3, m = 1 and gradually weaken as the size of the (mG/C)n block decreases (with a concomitant increase of (A/T)m). (A/T)2 nearest neighbor flank preferentially encloses the (G/C)n block (to produce (A/T)2 (G/C)n (A/T)m). The (A/T)2 flank trends are weaker than the (G/C)2 flank ones. The (A/T)2 flank trends also decrease in strength as the size of the (G/C)n block decreases. The statistical significance of these trends in eukaryotes is very high. A possible correlation with DNA structural parameters, in particular groove geometry, is discussed.     

Ectopic expression of AID in a non-B cell line triggers A:T and G:C point mutations in non-replicating episomal vectors

Somatic hypermutation (SHM) of immunoglobulin genes is currently viewed as a two step process initiated by the deamination of deoxycytidine (C) to deoxyuridine (U), catalysed by the activation induced deaminase (AID). Phase 1 mutations arise from DNA replication across the uracil residue or the abasic site, generated by the uracil-DNA glycosylase, yielding transitions or transversions at G:C pairs. Phase 2 mutations result from the recognition of the U:G mismatch by the Msh2/Msh6 complex (MutS Homologue), followed by the excision of the mismatched nucleotide and the repair, by the low fidelity DNA polymerase eta, of the gap generated by the exonuclease I. These mutations are mainly focused at A:T pairs. Whereas in activated B cells both G:C and A:T pairs are equally targeted, ectopic expression of AID was shown to trigger only G:C mutations on a stably integrated reporter gene. Here we show that when using non-replicative episomal vectors containing a GFP gene, inactivated by the introduction of stop codons at various positions, a high level of EGFP positive cells was obtained after transient expression in Jurkat cells constitutively expressing AID. We show that mutations at G:C and A:T pairs are produced. EGFP positive cells are obtained in the absence of vector replication demonstrating that the mutations are dependent only on the mismatch repair (MMR) pathway. This implies that the generation of phase 1 mutations is not a prerequisite for the expression of phase 2 mutations.     

The exon A (C77G) mutation is a common cause of abnormal CD45 splicing in humans

The leukocyte common (CD45) Ag is essential for normal T lymphocyte function and alternative splicing at the N terminus of the gene is associated with changes in T cell maturation and differentiation. Recently, a statistically significant association was reported in a large series of human thymus samples between phenotypically abnormal CD45 splicing and the presence of the CC chemokine receptor 5 deletion 32 (CCR5del32) allele, which confers resistance to HIV infection in homozygotes. We show here that abnormal splicing in these thymus samples is associated with the presence of the only established cause of CD45 abnormal splicing, a C77G transversion in exon A. In addition we have examined 227 DNA samples from peripheral blood of healthy donors and find no association between the exon A (C77G) and CCR5del32 mutations. Among 135 PBMC samples, tested by flow cytometric analysis, all those exhibiting abnormal splicing of CD45 also showed the exon A C77G transversion. We conclude that the exon A (C77G) mutation is a common cause of abnormal CD45 splicing and that further disease association studies of this mutation are warranted.     

Hepatocarcinogen quinoline induces G:C to C:G transversions in the cII gene in the liver of lambda/lacZ transgenic mice (MutaMouse)

Quinoline is carcinogenic to the liver in rodents, but it is not clear whether it acts by a genotoxic mechanism. We previously demonstrated that quinoline does induce gene mutation in the liver of lambda/lacZ transgenic mice. In the present report, we reveal the molecular nature of the mutations induced by quinoline in the lambda cII gene, which is also a phenotypically selectable marker in the lambda transgene. (The cII gene has 294bp, which enables much easier sequence analysis than the original lacZ gene (3kb)). The liver cII mutant frequency was nine times higher in quinoline-treated mice than in control mice. Sequence analysis revealed that quinoline induced primarily G:C to C:G transversions (25 of 34). Thus, we have confirmed that quinoline is genotoxic in its target organ, and the G:C to C:G transversion is the molecular signature of quinoline-induced mutations.     

A DNA repair process in Escherichia coli corrects U:G and T:G mismatches to C:G at sites of cytosine methylation

Escherichia coli contains a base mismatch correction system called VSP repair that is known to correct T:G mismatches to C:G when they occur in certain sequence contexts. The preferred sequence context for this process is the site for methylation by the E. coli DNA cytosine methylase (Dcm). For this reason, VSP repair is thought to counteract potential mutagenic effects of deamination of 5-methylcytosine to thymine. We have developed a genetic reversion assay that quantitates the frequency of C to T mutations at Dcm sites and the removal of such mutations by DNA repair processes. Using this assay, we have studied the repair of U: G mismatches in DNA to C: G and have found that VSP repair is capable of correcting these mismatches. Although VSP repair substantially affects the reversion frequency, it may not be as efficient at correcting U: G mismatches as the uracil DNA glycosylase-mediated repair process.     

N-methyl-N'-nitro-N-nitrosoguanidine-induced mutation in a RecA strain of Escherichia coli

274 N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced forward mutations in the lacI gene of an Escherichia coli RecA- strain were cloned and sequenced. Base substitutions accounted for 264 mutations and consisted of 261 G:C----A:T transitions (including one double mutant with two G:C----A:T transitions separated by 25 base pairs), two A:T----G:C transitions and one A:T----T:A transversion. Therefore, 263 of the 274 mutations (all the transitions) can be explained as a result of the direct mispairing of O6-methylguanine, and O4-methylthymine residues during DNA synthesis. The source of the transversion is not known. The remaining mutations, one 16-base pair deletion, two -1 frameshifts and 7 frameshifts at the lacI frameshift hotspot, are located in runs of identical bases or flanked by directly repeated DNA sequences and can therefore be explained by template slippage events during DNA synthesis. The observed distribution of mutations recovered is identical to that found in a RecA+ background indicating little involvement of RecA function in MNNG-induced mutation. Analysis of neighbouring base sequence revealed that the G:C----A:T transition was 6 times more likely to be recovered if the mutated guanine residue was preceded by a purine rather than a pyrimidine. A most striking aspect of this distribution concerns particular residues in the core domain of the lac repressor protein. Within this domain the great majority of mutations generate nonsense codons or alter Gly codons.     

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.     

A role for DNA polymerase V in G --> T mutations from the major benzo[a]pyrene N2-dG adduct when studied in a 5'-TGT sequence in E. coli

Benzo[a]pyrene (B[a]P), a potent mutagen/carcinogen, is metabolically activated to (+)-anti-B[a]PDE, which induces a full spectrum of mutations (e.g. GC --> TA, GC --> AT, etc.) principally via its major adduct [+ta]-B[a]P-N2-dG. Recent findings suggest that different lesion bypass DNA polymerases may be involved in different mutagenic pathways, which is the subject of this report. [+ta]-B[a]P-N2-dG built into a plasmid in a 5'-TGT sequence gives approximately equal numbers of G --> T and G --> A mutations when host E. coli are UV irradiated prior to transformation, so this sequence context was chosen to investigate what DNA polymerases are involved in G --> T versus G --> A mutations. G --> T mutations decline (>10-fold) if E. coli either are not UV-irradiated or are deficient in DNA polymerase V ((delta)umuD/C), demonstrating a role for damage-inducible DNA Pol V in a G --> T pathway. G --> T mutations are not affected by transformation into E. coli deficient in either DNA polymerases II or IV. While the work herein was in progress, Lenne-Samuel et al. [Mol. Microbiol. 38 (2000) 299] built the same adduct into a plasmid in a 5'-GGA sequence, and showed that the frequency of G --> T mutations was similar in UV-irradiated and unirradiated host E. coli cells, suggesting no involvement by damage-inducible, lesion bypass DNA polymerases (i.e., not II, IV or V); furthermore, a role for DNA Pol V was explicitly ruled out. The easiest way to reconcile the findings of Lenne-Samuel et al. with the findings herein is if two G --> T mutagenic pathways exist for [+ta]-B[a]P-N2-dG, where sequence context dictates which pathway is followed. In contrast to the G --> T mutations, herein G --> A mutations from [+ta]-B[a]P-N2-dG in the 5'-TGT sequence context are shown not to be affected by UV-irradiation of host E. coli, and are not dependent on DNA Pol V, or Pol II, Pol IV, or the damage-inducible, but SOS-independent UVM system. Published studies, however, have shown that G --> A mutations are usually enhanced by UV-irradiation of host E. coli prior to the introduction of plasmids either site-specifically modified with [+ta]-B[a]P-N2-dG or randomly adducted with (+)-anti-B[a]PDE; both findings imply the involvement of a lesion-bypass DNA polymerase. These disparate results suggest the existence of two G --> A mutagenic pathways for [+ta]-B[a]P-N2-dG as well, although confirmation of this awaits further study. In conclusion, a comparison between the evidence presented herein and published findings suggests the existence of two distinct mutagenic pathways for both G --> T and G --> A mutations from [+ta]-B[a]P-N2-dG, where in each case one pathway is not damage-inducible and not dependent on a lesion-bypass DNA polymerase, while the second pathway is damage-inducible and dependent on a lesion-bypass DNA polymerase. Furthermore, DNA sequence context appears to dictate which pathway (as defined by the involvement of different DNA polymerases) is followed in each case.     

General Nearest Neighbor Preferences in G/C Oligomers Interrupted by A/T: Correlation with DNA Structure

Abstract

The frequencies of occurrence of the 5′ and 3′ nearest neighbor doublets of oligonucleotides containing (G/C) and (A/T) blocks show strong trends. Specifically, the following trends are observed. Given a (G/C)_n (A/T)_m oligomer (where G/C)_n indicates a sequence of length n composed solely of Gs and/or Cs and (A/T)_mis a sequence of length m composed solely of As and/or Ts, and n=3,2,1; m = 1,2,3) and a (G/C)₂ doublet, (G/C)_n (A/T)_m (G/C)_n > (G/C)_n+2 (A/T)_m- That is the (G/C)₂ doublet is preferentially located 3′ of the oligomer, enclosing the (A/T)_m stretch. The trends are strongest for n=3, m= 1 and gradually weaken as the size of the (G/C)_n block decreases (with a concomitant increase of (A/T)_m). (A/T)₂ nearest neighbor flank preferentially encloses the (G/C)_n block (to produce (A/T) ₂(G/C) _n (A/T)_m). The (A/T)₂ flank trends are weaker than the (G/C)₂ flank ones. The (A/T)₂ flank trends also decrease in strength as the size of the (G/C)_n block decreases. The statistical significance of these trends in eukaryotes is very high. A possible correlation with DNA structural parameters, in particular groove geometry, is discussed.     

Mutations induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the lacZ and cII genes of Muta™ Mouse

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) found in chewing tobacco, snuff, cigarettes, and cigars is a tobacco-specific nitrosamine and classified as a possible human carcinogen (Class 2B) by the International Agency for Research on Cancer (IARC). NNK given intraperitoneally was seen to induce lung and liver adenomas.To evaluate the genotoxicity of NNK in vivo, NNK was intraperitoneally administered to Muta™ Mouse at two concentrations (125 and 250 mg/kg, once a week for 4 weeks) followed by the measurement of mutant frequencies in the lacZ and cII genes from lung and liver in the same mice. Characterization of the types of the mutation was determined by sequencing the cII genes from mutant plaques. The mutant frequencies in both target genes from both organs dose-dependently increased up to 10 times compared to those of the control group. For the types of mutations, the ratio of the G:C to A:T mutation in the total number of mutants was less than the ratio of A:T to T:A and A:T to C:G transversion, contrary to a previous report [Cancer Res, 49 (1989) 5305]. The A:T to T:A transversion was the most highly induced mutation both in the lung and liver cII genes. The increasing rate of mutant frequencies in lung and liver over the vehicle control was 55 and 56 times, respectively, while the increasing rate of G:C to A:T transition was only 1.9 and 2.8 times, respectively.These observations show that NNK predominantly induces DNA adducts leading to A:T to T:A and/or A:T to C:G mutations in the transgene.