DNA damage-induced mutation: tolerance via translesion synthesis.

Translesion synthesis (TLS) appears to be required for most damage-induced mutagenesis in the yeast Saccharomyces cerevisiae, whether the damage arises from endogenous or exogenous sources. Thus, the production of such mutations seems to occur primarily as a consequence of the tolerance of DNA lesions rather than an error-prone repair mechanism. Tolerance via TLS in yeast involves proteins encoded by members of the RAD6 epistasis group for the repair of ultraviolet (UV) photoproducts, in particular two non-essential DNA polymerases that catalyse error-free or error-prone TLS. Homologues of these RAD6 group proteins have recently been discovered in rodent and/or human cells. Furthermore, the operation of error-free TLS in humans has been linked to a reduced risk of UV-induced skin cancer, whereas mutations generated by error-prone TLS may increase the risk of cancer. In this article, we review and link the evidence for translesion synthesis in yeast, and the involvement of nonreplicative DNA polymerases, to recent findings in mammalian cells.

Evidence from mutational specificity studies that yeast DNA polymerases delta and epsilon replicate different DNA strands at an intracellular replication fork.

Although polymerases delta and epsilon are required for DNA replication in eukaryotic cells, whether each polymerase functions on a separate template strand remains an open question. To begin examining the relative intracellular roles of the two polymerases, we used a plasmid-borne yeast tRNA gene and yeast strains that are mutators due to the elimination of proofreading by DNA polymerases delta or epsilon. Inversion of the tRNA gene to change the sequence of the leading and lagging strand templates altered the specificities of both mutator polymerases, but in opposite directions. That is, the specificity of the polymerase delta mutator with the tRNA gene in one orientation bore similarities to the specificity of the polymerase epsilon mutator with the tRNA gene in the other orientation, and vice versa. We also obtained results consistent with gene orientation having a minor influence on mismatch correction of replication errors occurring in a wild-type strain. However, the data suggest that neither this effect nor differential replication fidelity was responsible for the mutational specificity changes observed in the proofreading-deficient mutants upon gene inversion. Collectively, the data argue that polymerases delta and epsilon each encounter a different template sequence upon inversion of the tRNA gene, and so replicate opposite strands at the plasmid DNA replication fork.

Analysis of yeast pms1, msh2, and mlh1 mutators points to differences in mismatch correction efficiencies between prokaryotic and eukaryotic cells.

Genetic stability relies in part on the efficiency with which post-replicative mismatch repair (MMR) detects and corrects DNA replication errors. In Escherichia coli, endogenous transition mispairs and insertion/deletion (ID) heterologies are corrected with similar efficiencies--but much more efficiently than transversion mispairs--as revealed by mutation rate increases in MMR mutants. To assess the relative efficiencies with which these mismatches are corrected in the yeast Saccharomyces cerevisiae, we examined repair of defined mismatches on heteroduplex plasmids and compared the spectra for >1000 spontaneous SUP4-o mutations arising in isogenic wild-type or MMR-deficient (pms1, mlh1, msh2) strains. Heteroduplexes containing G/T mispairs or ID heterologies were corrected more efficiently than those containing transversion mismatches. However, the rates of single base-pair insertion/deletion were increased much more (82-fold or 34-fold, respectively) on average than the rate of base pair substitutions (4.4-fold), with the rates for total transitions and transversions increasing to similar extents. Thus, the relative efficiencies with which mismatches formed during DNA replication are repaired appear to differ in prokaryotic and eukaryotic cells. In addition, our results indicate that in yeast, and probably other eukaryotes, these efficiencies may not mirror those obtained from an analysis of heteroduplex correction.

Strand interruptions confer strand preference during intracellular correction of a plasmid-borne mismatch in Saccharomyces cerevisiae.

Site-directed mutagenesis was used to construct yeast centromere plasmids in which a strand nick or gap could be placed 5' or 3', on either strand, to a reporter gene (SUP4-o) carrying defined base mismatches. The plasmids were then transformed into yeast cells and the direction and efficiency of mismatch repair were assayed by scoring colouring of the transformant colonies. Strands that were nicked were consistently corrected more often than intact strands, but the effect was very small. However, placement of a small gap at the same positions as the nicks resulted in a marked increase in selection for the gapped strand and an enhanced efficiency of mismatch repair. Both the preference for the gapped strand and correction of the mismatch were offset by deletion of the mismatch repair gene PMS1. Together, the results suggest that strand interruptions can direct intracellular mismatch correction of plasmid-borne base mispairs in yeast.

Defects in base excision repair combined with elevated intracellular dCTP levels dramatically reduce mutation induction in yeast by ethyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine.

Previously, we determined that elimination of deoxycytidylate (dCMP) deaminase (DCD1) in the yeast Saccharomyces cerevisiae increases the intracellular dCTP:dTTP ratio and reduces the induction of G x C --> A x T transitions in the SUP4-o gene by ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Simultaneously, the G x C --> C x G transversion frequency rises substantially. We attributed the first response to dCTP outcompeting dTTP for incorporation opposite O6-alkylguanine, and the second outcome to the increased dCTP pool causing error-prone repair of apurinic (AP) sites resulting from the removal or lability of N7-alkylguanine. To test the latter hypothesis, we used isogenic dcd1 strains deleted for either of two genes (MAG1: 3-methyladenine glycosylase; APN1: apurinic endonuclease) involved in the repair of N7-alkylguanine. In these backgrounds, EMS or MNNG induction of total SUP4-o mutations, G x C --> A x T transitions and G x C --> C x G transversions were reduced by >98%, >97%, and >80%, respectively. Mutation frequencies in the dcd1 apn1 strain were close to those for spontaneous mutagenesis in the wild-type parent. These findings argue that misincorporation of dCTP during repair of alkylation-induced AP sites is responsible for the increased G x C --> C x G transversion frequency in the dcd1 strain treated with EMS or MNNG. The data also demonstrate that defective repair of AP sites coupled with an elevated dCTP:dTTP ratio eliminates most EMS and MNNG mutagenesis. In addition, the results point to a role for AP sites in the production of some EMS- and MNNG-induced G x C --> A x T transitions as well as other substitutions in the dcd1 strain.

DNA repair in higher plants.

Numerous studies have demonstrated a requirement in plants for repair of DNA damage arising from either intrinsic or extrinsic sources. Investigations also have revealed a capacity for repair of certain types of DNA damage, and conversely, identified mutants apparently defective in such repair. This article provides a concise overview of nuclear DNA repair mechanisms in higher plants, particularly those processes concerned with the repair of UV-induced lesions, and includes surveys of UV-sensitive mutants and genes implicated in DNA repair.

DNA sequence analysis of spontaneous mutagenesis in Saccharomyces cerevisiae.

To help elucidate the mechanisms involved in spontaneous mutagenesis, DNA sequencing has been applied to characterize the types of mutation whose rates are increased or decreased in mutator or antimutator strains, respectively. Increased spontaneous mutation rates point to malfunctions in genes that normally act to reduce spontaneous mutation, whereas decreased rates are associated with defects in genes whose products are necessary for spontaneous mutagenesis. In this article, we survey and discuss the mutational specificities conferred by mutator and antimutator genes in the budding yeast Saccharomyces cerevisiae. The implications of selected aspects of the data are considered with respect to the mechanisms of spontaneous mutagenesis.

Differences in the mutational specificities of sunlight and UVB radiation suggest a role for transversion-inducing DNA damage in solar photocarcinogenesis.

Mutations induced by UVB radiation and natural sunlight in a plasmid-borne yeast (Saccharomyces cerevisiae) tRNA gene (SUP4-o) were characterised by DNA sequencing. For both agents, the majority (> 90%) of the total mutations analysed were single base-pair substitutions, but tandem substitutions and single base-pair deletions also were detected. Each agent induced all six types of base-pair change but the tandem substitutions involved exclusively G.C-->A.T transitions. However, the fractions of single and tandem G.C-->A.T transitions were reduced by about 50%, and the fraction of transversions at G.C pairs was increased by 11-fold for sunlight relative to UVB. Comparisons of the site and strand specificities of the substitutions suggested that dipyrimidine adducts were responsible for the transitions, and that other lesions induced by sunlight may have given rise to the transversions. The relevance of these findings to skin cancer is discussed.

A mutation in a Saccharomyces cerevisiae gene (RAD3) required for nucleotide excision repair and transcription increases the efficiency of mismatch correction.

RAD3 functions in DNA repair and transcription in Saccharomyces cerevisiae and particular rad3 alleles confer a mutator phenotype, possibly as a consequence of defective mismatch correction. We assessed the potential involvement of the Rad3 protein in mismatch correction by comparing heteroduplex repair in isogenic rad3-1 and wild-type strains. The rad3-1 allele increased the spontaneous mutation rate but did not prevent heteroduplex repair or bias its directionality. Instead, the efficiency of mismatch correction was enhanced in the rad3-1 strain. This surprising result prompted us to examine expression of yeast mismatch repair genes. We determined that MSH2, but not MLH1, is transcriptionally regulated during the cell-cycle like PMS1, and that rad3-1 does not increase the transcript levels for these genes in log phase cells. These observations suggest that the rad3-1 mutation gives rise to an enhanced efficiency of mismatch correction via a process that does not involve transcriptional regulation of mismatch repair. Interestingly, mismatch repair also was more efficient when error-editing by yeast DNA polymerase delta was eliminated. We discuss our results in relation to possible mechanisms that may link the rad3-1 mutation to mismatch correction efficiency.

Inhibitors of thymine nucleotide biosynthesis: antimetabolites that provoke genetic change via primary non-DNA targets.

Folate antagonists and direct-acting inhibitors of thymidylate synthase are potent genotoxic antimetabolites. These agents induce genetic change not by attacking DNA, but by interfering with the control of DNA precursor metabolism. This review surveys the genetic effects attributable to selected representatives of this class of antimetabolites.

Specificities of the Saccharomyces cerevisiae rad6, rad18, and rad52 mutators exhibit different degrees of dependence on the REV3 gene product, a putative nonessential DNA polymerase.

The Saccharomyces cerevisiae rad6, rad18, and rad52 mutants exhibit DNA repair deficiencies and distinct mutator phenotypes. DNA replication past unrepaired spontaneous damage might contribute to the specificities of these mutators. Because REV3 is thought to encode a DNA polymerase that specializes in translesion synthesis, we determined the REV3 dependence of the rad mutator specificities. Spontaneous mutagenesis at a plasmid-borne SUP4-o locus was examined in isogenic strains having combinations of normal or mutant REV3 and RAD6, RAD18, or RAD52 alleles. For the rad6 and rad18 mutators, the mutation rate increase relied largely, but not exclusively, on REV3 whereas the rad52 mutator was entirely REV3 dependent. The influence of REV3 on the specificity of the rad6 mutator differed markedly depending on the mutational class examined. However, the requirement of rev3 for the production of G.C-->T.A transversions by the rad18 mutator, which induces only these substitutions, was similar to that for rad6-mediated G.C-->T.A transversion. This supports a role for the Rad6-Rad18 protein complex in the control of spontaneous mutagenesis. The available data imply that the putative Rev3 polymerase can process a variety of spontaneous DNA lesions that normally are substrates for error-free repair.

Failure to detect an antimutator phenotype following disruption of the Saccharomyces cerevisiae DDR48 gene.

The antimutator phenotype, reportedly conferred by disruption of the Saccharomyces cerevisiae DDR48 gene, was suggested to affect only a specific spontaneous mutational pathway. We attempted to identify the types of mutation that are DDR48-dependent by determining the specificity of the ddr48 antimutator. However, disruption of DDR48 did not decrease the rates of spontaneous forward mutation in a plasmid-borne copy of the yeast SUP4-o gene, the reversion or suppression of the lys2-1 allele, or forward mutation at the CAN1 locus. Interestingly, the latter gene had been reported previously to be subject to the antimutator effect. DNA sequence analysis of spontaneous SUP4-o mutations arising in DDR48 and ddr48 backgrounds provided no evidence for a reduction in the rates of individual mutational classes. Thus, we were unable to verify that disruption of DDR48 causes an antimutator phenotype.

Excision repair and gene orientation modulate the strand specificity of UV mutagenesis in a plasmid-borne yeast tRNA gene.

Ultraviolet (UV) mutagenesis in a plasmid-borne Saccharomyces cerevisiae tRNA gene (SUP4-o) occurs preferentially at sites where the pyrimidine in the base pair is part of a dipyrimidine sequence on the transcribed strand. In this study, we examined whether excision repair, or strand identity with respect to DNA replication, influences this strand bias. The specificity of UV mutagenesis was determined for a wild type (RAD) strain and an isogenic excision repair-deficient (rad1) derivative, each carrying SUP4-o on the vector YCpMP2, or another vector (YCpJA1) that differed only in the orientation of SUP4-o with respect to a unique origin of replication. Most (> or = 90%) of the SUP4-o mutations induced by UV in these strains were single base pair substitutions, predominantly (> 87%) transitions. The rad1 defect and inversion of SUP4-o in the RAD strain eliminated the strand preference, whereas inversion of SUP4-o in the rad1 strain caused it to reappear. Both conditions also altered the distribution of frequently mutated sites and the relative fraction of transitions at TT sequences. These results suggest that excision repair and gene orientation can be important determinants for the strand and site specificities of UV mutagenesis in SUP4-o on YCpMP2 and YCpJA1. We consider several possible explanations for our observations, including potential roles for transcription by RNA polymerase II, sequence context effects on the efficiency of excision repair, and inherent differences in strand mutability or translesion synthesis by the leading and lagging strand DNA replication complexes.

Roles for the yeast RAD18 and RAD52 DNA repair genes in UV mutagenesis.

Experimental evidence indicates that although the Saccharomyces cerevisiae RAD18 and RAD52 genes are not required for nucleotide excision repair, they function in the processing of UV-induced DNA damage in yeast. Conflicting statements regarding the UV mutability of strains deleted for RAD18 prompted us to re-examine the influence of RAD18, and RAD52, on UV mutagenesis. To do so, we characterized mutations induced by UV in SUP4-o, a yeast suppressor tRNA gene. SUP4-o was maintained on a plasmid in isogenic strains that either carried one of two different rad18 deletions (rad18 delta) or had RAD52 disrupted. Both rad18 deletions decreased the frequency of UV-induced SUP4-o mutations to levels close to those for spontaneous mutagenesis in the rad18 delta backgrounds, and prevented a net increase in mutant yield. A detailed analysis of mutations isolated after UV irradiation of one of the rad18 delta strains uncovered little evidence of the specificity features typical for UV mutagenesis in the isogenic repair-proficient (RAD) parent (e.g., predominance of G.C-->A.T transitions). Evidently, UV induction of SUP4-o mutations is highly dependent on the RAD18 gene. Compared to the RAD strain, disruption of RAD52 reduced the frequency and yield of UV mutagenesis by about two-thirds. Closer inspection revealed that 80% of this reduction was due to a decrease in the frequency of G.C-->A.T transitions. In addition, there were differences in the distributions and site specificities of single base-pair substitutions. Thus, RAD52 also participates in UV mutagenesis of a plasmid-borne gene in yeast, but to a lesser extent than RAD18.

International Commission for Protection Against Environmental Mutagens and Carcinogens. Deoxyribonucleoside triphosphate levels: a critical factor in the maintenance of genetic stability.

DNA precursor pool imbalances can elicit a variety of genetic effects and modulate the genotoxicity of certain DNA-damaging agents. These and other observations indicate that the control of DNA precursor concentrations is essential for the maintenance of genetic stability, and suggest that factors which offset this control may contribute to environmental mutagenesis and carcinogenesis. In this article, we review the biochemical and genetic mechanisms responsible for regulating the production and relative amounts of intracellular DNA precursors, describe the many outcomes of perturbations in DNA precursor levels, and discuss implications of such imbalances for sensitivity to DNA-damaging agents, population monitoring, and human diseases.

Specificity of the mutator caused by deletion of the yeast structural gene (APN1) for the major apurinic endonuclease.

The loss of bases from cellular DNA occurs via both spontaneous and mutagen-induced reactions. The resulting apurinic/apyrimidinic (AP) sites are cytotoxic and mutagenic but are counteracted by repair initiated by AP endonucleases. Previously, in vitro and bacterial transfection studies suggested that AP sites often prompt insertion of dAMP residues during replication, the A-rule. Dissimilar results have been obtained by transfecting DNA into eukaryotic cells. It seemed possible that these differences might be due to idiosyncrasies of transfection or aberrant replication of the transecting DNA. The observation that AP endonuclease-deficient strains of the yeast Saccharomyces cerevisiae have elevated spontaneous mutation rates allowed us to determine the mutational specificity of endogenously generated AP sites in nuclear DNA. With the yeast SUP4-o gene as a mutational target, we found that a deficiency in the major yeast AP endonuclease, Apn1, provoked mainly single base-pair substitution; the rate of transposon Ty insertion was also enhanced. The rate of transversion to a G.C pair was increased 10-fold in Apn1-deficient yeast, including a 59-fold increase in the rate of A.T-->C.G events. In contrast, the rate of transversion to an A.T pair was increased by only 3-fold. A deficiency in N3-methyladenine glycosylase offset these substitution rate increases, indicating that they are due primarily to AP sites resulting from glycosylase action. Thus, the A-rule does not seem to apply to the mutagenic processing of endogenous abasic sites in S. cerevisiae. Other results presented here show that AP endonuclease-deficient Escherichia coli exhibit a mutator phenotype consistent with the A-rule.

Specificity of the yeast rev3 delta antimutator and REV3 dependency of the mutator resulting from a defect (rad1 delta) in nucleotide excision repair.

The yeast REV3 gene has been predicted to encode a DNA polymerase specializing in translesion synthesis. This polymerase likely participates in spontaneous mutagenesis, as rev3 mutants have an antimutator phenotype. Translesion synthesis also may be necessary for the mutator caused by a RAD1 (nucleotide excision repair) deletion (rad1 delta). To further examine the role of REV3 in spontaneous mutagenesis, we characterized SUP4-o mutations that arose spontaneously in strains having combinations of normal or mutant REV3 and RAD1 alleles. The largest fraction of the rev3 delta-dependent mutation rate decrease was observed for single base-pair substitutions and deletions, although the rates of all mutational classes detected in the RAD1 background were reduced by at least 30%. Interestingly, inactivation of REV3 was associated with a doubling of the number of sites at which the retrotransposon Ty inserted. rev3 delta also greatly diminished the magnitude of the rad1 delta mutator, but not to the rev3 delta antimutator level, implicating REV3-dependent and independent processes in the rad1 delta mutator effect. However, the specificity of the rev3 delta antimutator suggested that the same REV3-dependent processes gave rise to the majority of spontaneous mutations in the RAD1 and rad1 delta strains.

Elevated intracellular dCTP levels reduce the induction of GC-->AT transitions in yeast by ethyl methanesulfonate or N-methyl-N'-nitro-N- nitrosoguanidine but increase alkylation-induced GC-->CG transversions.

The effect of an increased intracellular dCTP:dTTP ratio on the specificities of ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis was examined in the yeast Saccharomyces cerevisiae. To do so, we used a dCMP deaminase-deficient (dcd1) strain having a dCTP:dTTP ratio > 77-fold larger than its isogenic wild-type parent under the treatment conditions employed. This DNA precursor imbalance lowered the frequencies of EMS- or MNNG-induced SUP4-o mutations by 75 or 45%, respectively, relative to the corresponding values for the wild-type strain. A total of 405 SUP4-o mutations produced by the alkylating agents in the dcd1 background were characterized by DNA sequencing and the mutational spectra were compared to those for 399 mutations induced in the wild-type parent and 207 mutations that arose spontaneously in the dcd1 strain. Unexpectedly, the frequencies of EMS- and MNNG-induced GC-->AT transitions in the dcd1 strain were found to be reduced by 93 and 68%, respectively, considerably more than the decreases for the overall SUP4-o mutation frequencies. The differences were due mainly to substantial increases in the frequencies of GC-->CG transversions. Although these events were the predominant type of spontaneous substitution in the dcd1 strain, they were more frequent after alkylation treatment and were distributed differently than the spontaneous GC-->CG transversions. Preferences for the EMS- or MNNG-induced GC-->AT transitions to occur at GC sites having the guanine located on the transcribed strand or preceded by a 5' purine, respectively, also were diminished in the dcd1 strain.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutational specificity of thymine nucleotide depletion in yeast.

Relative to normal growth conditions for a wild-type strain of the yeast Saccharomyces cerevisiae, withholding thymidylate (dTMP) severely diminished the dTTP pool but elevated the dATP, dGTP and dCTP levels (120-, 8.5- and 3.6-fold, respectively) in an isogenic dTMP auxotroph. This treatment also increased the frequency of mutations in a tRNA gene (SUP4-o) by 15-fold. Single base-pair events accounted for 97% of the 89 SUP4-o mutations characterized by DNA sequencing and the ratio of transversions to transitions was 3-fold greater than that for spontaneous substitutions in the wild-type strain. This difference was due to decreases in the fractions of transitions and an increase in the proportion of A.T-->T.A transversions. The largest increases in mutation frequency were observed for transversions at A.T pairs, consistent with dATP and dGTP being incorporated in place of dTTP during DNA replication. Similarly, misinsertion of dATP and dGTP could have promoted the single base-pair deletions and insertion detected. Analysis of the distributions of substitutions indicated no preference for dATP misinsertion to occur at sites flanked by a specific 5' or 3' base or on the transcribed or nontranscribed strands. However, the presence of mutational hotspots and site-specific variations in the substitution frequencies implied a role for DNA sequence context in the mutational specificity of dTTP depletion.