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.     

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

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

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.     

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.     

The mutT defect does not elevate chromosomal fragmentation in Escherichia coli because of the surprisingly low levels of MutM/MutY-recognized DNA modifications

Nucleotide pool sanitizing enzymes Dut (dUTPase), RdgB (dITPase), and MutT (8-oxo-dGTPase) of Escherichia coli hydrolyze noncanonical DNA precursors to prevent incorporation of base analogs into DNA. Previous studies reported dramatic AT-->CG mutagenesis in mutT mutants, suggesting a considerable density of 8-oxo-G in DNA that should cause frequent excision and chromosomal fragmentation, irreparable in the absence of RecBCD-catalyzed repair and similar to the lethality of dut recBC and rdgB recBC double mutants. In contrast, we found mutT recBC double mutants viable with no signs of chromosomal fragmentation. Overproduction of the MutM and MutY DNA glycosylases, both acting on DNA containing 8-oxo-G, still yields no lethality in mutT recBC double mutants. Plasmid DNA, extracted from mutT mutM double mutant cells and treated with MutM in vitro, shows no increased relaxation, indicating no additional 8-oxo-G modifications. Our DeltamutT allele elevates the AT-->CG transversion rate 27,000-fold, consistent with published reports. However, the rate of AT-->CG transversions in our mutT(+) progenitor strain is some two orders of magnitude lower than in previous studies, which lowers the absolute rate of mutagenesis in DeltamutT derivatives, translating into less than four 8-oxo-G modifications per genome equivalent, which is too low to cause the expected effects. Introduction of various additional mutations in the DeltamutT strain or treatment with oxidative agents failed to increase the mutagenesis even twofold. We conclude that, in contrast to the previous studies, there is not enough 8-oxo-G in the DNA of mutT mutants to cause elevated excision repair that would trigger chromosomal fragmentation.     

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.     

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

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

The 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.     

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.     

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.     

Antimutator role of DNA glycosylase MutY in pathogenic Neisseria species

Genome alterations due to horizontal gene transfer and stress constantly generate strain on the gene pool of Neisseria meningitidis, the causative agent of meningococcal (MC) disease. The DNA glycosylase MutY of the base excision repair pathway is involved in the protection against oxidative stress. MC MutY expressed in Escherichia coli exhibited base excision activity towards DNA substrates containing A:7,8-dihydro-8-oxo-2'-deoxyguanosine and A:C mismatches. Expression in E. coli fully suppressed the elevated spontaneous mutation rate found in the E. coli mutY mutant. An assessment of MutY activity in lysates of neisserial wild-type and mutY mutant strains showed that both MC and gonococcal (GC) MutY is expressed and active in vivo. Strikingly, MC and GC mutY mutants exhibited 60- to 140-fold and 20-fold increases in mutation rates, respectively, compared to the wild-type strains. Moreover, the differences in transitions and transversions in rpoB conferring rifampin resistance observed with the wild type and mutants demonstrated that the neisserial MutY enzyme works in preventing GC-->AT transversions. These findings are important in the context of models linking mutator phenotypes of disease isolates to microbial fitness.     

Studies on the replication of the ring opened formamidopyrimidine, Fapy.dG in Escherichia coli

Fapy.dG is produced in DNA as a result of oxidative stress from a precursor that also forms OxodG. Bypass of Fapy.dG in a shuttle vector in COS-7 cells produces G --> T transversions slightly more frequently than does OxodG (Kalam, M. A., et al. (2006) Nucleic Acids Res. 34, 2305). The effect of Fapy.dG on replication in Escherichia coli was studied by transfecting M13mp7(L2) bacteriophage DNA containing the lesion within the lacZ gene in 4 local sequence contexts. For comparison, experiments were carried out side-by-side on OxodG. The efficiency of lesion bypass was determined relative to that of a genome containing native nucleotides. Fapy.dG was bypassed less efficiently than OxodG. Bypass efficiency of Fapy.dG and OxodG increased modestly in SOS-induced cells. Mutation frequencies at the site of the lesions in the originally transfected genomes were determined using the REAP assay (Delaney, J. C., Essigmann, J. M. (2006) Methods Enzymol. 408, 1). G --> T transversions were the only mutations observed above background when either Fapy.dG or OxodG was bypassed. OxodG mutation frequencies ranged from 3.1% to 9.8%, whereas the G --> T transversion frequencies observed upon Fapy.dG bypass were T transversions.     

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.     

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.     

Development of antibiotic resistance and up-regulation of the antimutator gene pfpI in mutator Pseudomonas aeruginosa due to inactivation of two DNA oxidative repair genes (mutY, mutM)

Prevention and correction of oxidative DNA lesions in Pseudomonas aeruginosa is ensured by the DNA oxidative repair system (GO). Single inactivation of mutT, mutY and mutM involved in GO led to elevated mutation rates (MRs) that correlated to increased development of resistance to antibiotics. In this study, we constructed a double mutant in mutY and mutM (PAOMY-Mgm) and characterized the phenotype and the gene expression profile using microarray and RT-PCR. PAOMY-Mgm presented 28-fold increases in MR compared with wild-type reference strain PAO1. In comparison, the PAOMYgm (mutY) single mutant showed only a fivefold increase, whereas the single mutant PAOMMgm (mutM) showed a nonsignificant increase in MR compared with PAO1 and the single mutants. Mutations in the regulator nfxB leading to hyperexpression of MexCD-OprJ efflux pump were found as the mechanism of resistance to ciprofloxacin in the double mutant. A better fitness of the mutator compared with PAO1 was found in growth competition experiments in the presence of ciprofloxacin at concentrations just below minimal inhibitory concentration. Up-regulation of the antimutator gene pfpI, that has been shown to provide protection to oxidative stress, was found in PAOMY-Mgm compared with PAO1. In conclusion, we showed that MutY and MutM are cooperating in the GO of P. aeruginosa, and that oxidative DNA lesions might represent an oxidative stress for the bacteria.     

DNA alkylation repair limits spontaneous base substitution mutations in Escherichia coli.

The Escherichia coli Ada and Ogt DNA methyltransferases (MTases) are known to transfer simple alkyl groups from O6-alkylguanine and O4-alkylthymine, directly restoring these alkylated DNA lesions to guanine and thymine. In addition to being exquisitely sensitive to the mutagenic effects of methylating agents, E. coli ada ogt null mutants display a higher spontaneous mutation rate than the wild type. Here, we determined which base substitution mutations are elevated in the MTase-deficient cells by monitoring the reversion of six mutated lacZ alleles that revert via each of the six possible base substitution mutations. During exponential growth, the spontaneous rate of G:C to A:T transitions and G:C to C:G transversions was elevated about fourfold in ada ogt double mutant versus wild-type E. coli. Furthermore, compared with the wild type, stationary populations of the MTase-deficient E. coli (under lactose selection) displayed increased G:C to A:T and A:T to G:C transitions (10- and 3-fold, respectively) and increased G:C to C:G, A:T to C:G, and A:T to T:A transversions (10-, 2.5-, and 1.7-fold, respectively). ada and ogt single mutants did not suffer elevated spontaneous mutation rates for any base substitution event, and the cloned ada and ogt genes each restored wild-type spontaneous mutation rates to the ada ogt MTase-deficient strains. We infer that both the Ada MTase and the Ogt MTase can repair the endogenously produced DNA lesions responsible for each of the five base substitution events that are elevated in MTase-deficient cells. Simple methylating and ethylating agents induced G:C to A:T and A:T to G:C transitions in these strains but did not significantly induce G:C to C:G, A:T to C:G, and A:T to T:A transversions. We deduce that S-adenosylmethionine (known to e a weak methylating agent) is not the only metabolite responsible for endogenous DNA alkylation and that at least some of the endogenous metabolites that cause O-alkyl DNA damage in E. coli are not simple methylating or ethylating agents.     

DNA sequence analysis of spontaneous and γ-radiation (anoxic)-induced lacI mutations in Escherichia coliumuC122::Tn5: differential requirement for umuC at G · C vs. A · T sites and for the production of transversions vs. transitions

Escherihica coliumC122::Tn5 cells were γ-radiated (¹³⁷Cs, 750 Gy, under N₂), and lac-constitutive mutants were produced at 36% of the wild-type level (the umC strain was not deficient in spontaneous mutagenesis, and the mutational spectrum determined by sequencing 263 spontaneous lacI^d mutations was very similar to that for the wild-type strain). The specific nature of the umC strain's partial radiation was determined by sequencing 325 radiation-induced lacI^d mutations. The yields of radiation-induced mutation classes in the umC strain (as a percentage of the wild-type yield) were: 80% for A · T → G · C transitions, 70% for multi-base additions, 60% for single-base deletions, 53% for A · T → C · G transversions, 36% for G · C → A · T transitions, 25% for multi-base deletions, 21% for A · T → T · A transversions, 11% for G · C → C · G transversions, 9% for G · C → T · A transversions and 0% for multiple mutations. Based on these deficiencies and other factors, it is concluded that the umC strain is near-normal for A · T → G · C transitions, single-base deletions and possibly A · T → C · G transversions; is generally deficient for mutagenesis at G · C sites fro transversions, and is grossly deficient in multiple mutations. Damage at G · C sites seems more difficult for translesion DNA synthesis to bypass than damage at A · T sites, and especially when trying to produced a transversion. The yield of G · C → A · T transitions in the umC strain *36% of the wild-type level) argues that a basic sites are involved in no more than 64% of γ-radiation-induced base substitutions in the wild-type strain. Altogether, these data suggest that the UmuC and UmuD′ proteins facilitate, rather than being absolutely required for, translesion DNA synthesis; with the degree of facilitation being dependent both on the nature of the noncoding DNA damage, i.e., at G · C vs A · T sites, and on the nature of the misincorporated base, i.e., whether it induces transversions or transitions.     

The influence of combined Fpg- and MutY-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum in the lacZalpha gene of m13mp10

One of the most predominating oxidative DNA damages, both spontaneously formed and after gamma-radiation is 7, 8-dihydro-8-oxoguanine (8oxoG). This 8oxoG is a mutagenic lesion because it can mispair with adenine instead of the correct cytosine leading to G:C to T:A transversions. In Escherichia coli (E. Coli) base excision repair (BER) is one of the most important repair systems for the repair of 8oxoG and other oxidative DNA damage. An important part of BER in E. coli is the so-called GO system which consists of three repair enzymes, MutM (Fpg), MutY and MutT which are all involved in repair of 8oxoG or 8oxoG mispairs. The aim of this study is to determine the effect of combined Fpg- and MutY-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum of the lacZalpha gene. For that purpose, non-irradiated or gamma-irradiated double-stranded (ds) M13mp10 DNA, with the lacZalpha gene inserted as mutational target sequence was transfected into an E. coli strain which is deficient in both Fpg and MutY (BH1040). The resulting mutation spectra were compared with the mutation spectra of a fpg(-) E. coli strain (BH410) and a wild type E. coli strain (JM105) which were determined in an earlier study. The results of the present study indicate that combined Fpg- and MutY-deficiency induces a large increase in G:C to T:A transversions in both the spontaneous and gamma-radiation-induced mutation spectra of BH1040 (fpg(-)mutY(-)) as compared to the fpg(-) and the wild type strain. Besides the increased levels of G:C to T:A transversions, there is also an increase in G:C to C:G transversions and frameshift mutations in both the spontaneous and gamma-radiation-induced mutation spectra of BH1040 (fpg(-)mutY(-)).     

离子注入诱变莲花突变体分子机理的初步研究

低能离子注入技术作为生物物理诱变的一种新型技术, 在园艺植物育种方面具有很大的应用潜力, 但其诱变的分子机理目前知之甚少。文章对Fe+ 离子注入诱变的白洋淀红莲(Nelumbium speciosum Willd)突变体及其对照的基因组进行RAPD研究, 并将突变体和对照在辐射敏感位点的条带进行克隆测序及DNA序列分析。在已优化好的RAPD体系下扩增, 从110条随机引物中筛选出了10条可以稳定扩增出显著特异条带的引物, 引物多态性为9.09%。将这10条引物扩增出的辐射敏感位点的条带进行克隆测序, 并进行序列比对。结果显示: 突变体的总碱基突变频率为0.87%, 6个突变体的碱基突变频率存在着差异; 碱基突变类型包括碱基的颠换、转换、缺失、插入, 在检测到的159个碱基突变中, 单碱基置换的频率(61.01%)高于碱基插入或者缺失的频率(38.99%), 在碱基置换中, 转换的频率(44.65%)是颠换频率(16.35%)的2.7倍, 其中C/T之间的转换所占比例最大, A→G和A→T也具有较高的替换频率; 构成DNA的4种碱基均可以被离子束辐照诱变发生变异, 除了没有C→G的置换外, 每一种碱基都可以被其他的几种碱基所置换, 但是胸腺嘧啶(T)具有较高的辐射敏感性。通过对碱基突变位点周边序列的分析发现, 嘌呤突变位点的周围嘌呤碱居多, 嘧啶突变位点的周围嘧啶碱居多。研究结果为揭示低能离子注入诱变作用分子机理提供了依据。