Ultraviolet-Induced Reversion of cyc1 Alleles in Radiation Sensitive Strains of Yeast. II. rev2 Mutant Strains

The range of specificity of the rev2-1 mutation, an allele that reduces the frequency of ochre revertants induced by UV in Saccharomyces cerevisiae (LEMONTT 1971a), has been investigated by examining its influence on the reversion of eleven well-defined and contrasting cyc1 mutations. We have shown, in support of a suggestion of LEMONTT (1971a), that the REV2 gene product is concerned only with the reversion of ochre alleles; it plays virtually no role in the reversion of amber, missense or frameshift mutations. We have also shown that its effect is specific and confined to only some highly revertible ochre alleles. The REV2 gene product appears to enhance reversion at these sites by facilitating the conversion of two otherwise nonmutagenic photo-products into a single premutational lesion. UV-induced killing of rev2-1 strains was found to be significantly greater on fermentable rather than on nonfermentable media.     

Mutagenesis by Cytostatic Alkylating Agents in Yeast Strains of Differing Repair Capacities

Reversion of two nulcear ochre nonsense alleles and cell inactivation induced by mono-, bi-, and tri-functional alkylating agents and by UV has been investigated in stationary-phase haploid cells of yeast strains with differing capacities for DNA repair. The ability to survive alkylation damage is correlated with UV repair capacity, a UV-resistant and UV-mutable strain (RAD REV) being least and a UV-sensitive and UV-nonmutable strain (radi rev3) most sensitive. Mutagenicity of alkylating agents is highest in the former and is abolished in the latter strain. Deficiency in excision repair (rad1 rad2) or in the RAD18 function does not lead to enhanced mutability. Mutagenesis by the various agents is characterized by a common pattern of induction of locus-specific revertants and suppressor mutants. Induction kinetics are mostly linear, but UV-induced reversion in the RAD REV strain follows higher-than-linear (probably "quadratic") kinetics. The alkylating agent cyclophosphamide, usually considered inactive without metabolic conversion, reduces colony-forming ability and induces revertants in a manner similar but not identical to the other chemicals tested. These findings are taken to support the concept of mutagenesis by misrepair after alkylation, which albeit sharing common features with the mechanism of UV-induced reversion, can be distinguished therefrom.     

Alteration of ultraviolet-induced mutagenesis in yeast through molecular modulation of the REV3 and REV7 gene expression

DNA damage can lead to mutations during replication. The damage-induced mutagenesis pathway is an important mechanism that fixes DNA lesions into mutations. DNA polymerase zeta (Pol zeta), formed by Rev3 and Rev7 protein complex, and Rev1 are components of the damage-induced mutagenesis pathway. Since mutagenesis is an important factor during the initiation and progression of human cancer, we postulate that this mutagenesis pathway may provide an inhibiting target for cancer prevention and therapy. In this study, we tested if UV-induced mutagenesis can be altered by molecular modulation of Rev3 enzyme levels using the yeast Saccharomyces cerevisiae as a eukaryotic model system. Reducing the REV3 expression in yeast cells through molecular techniques was employed to mimic Pol zeta inhibition. Lower levels of Pol zeta significantly decreased UV-induced mutation frequency, thus achieving inhibition of mutagenesis. In contrast, elevating the Pol zeta level by enhanced expression of both REV3 and REV7 genes led to a approximately 3-fold increase in UV-induced mutagenesis as determined by the arg4-17 mutation reversion assays. In vivo, UV lesion bypass by Pol zeta requires the Rev1 protein. Even overexpression of Pol zeta could not alleviate the defective UV mutagenesis in the rev1 mutant cells. These observations provide evidence that the mutagenesis pathway could be used as a target for inhibiting damage-induced mutations.     

Mutator alleles of yeast DNA polymerase zeta

The yeast REV3 gene encodes the catalytic subunit of DNA polymerase zeta (pol zeta), a B family polymerase that performs mutagenic DNA synthesis in cells. To probe pol zeta mutagenic functions, we generated six mutator alleles of REV3 with amino acid replacements for Leu979, a highly conserved residue inferred to be at the pol zeta active site. Replacing Leu979 with Gly, Val, Asn, Lys, Met or Phe resulted in yeast strains with elevated UV-induced mutant frequencies. While four of these strains had reduced survival following UV irradiation, the rev3-L979F and rev3-L979M strains had normal survival, suggesting retention of pol zeta catalytic activity. UV mutagenesis in the rev3-L979F background was increased when photoproduct bypass by pol eta was eliminated by deletion of RAD30. The rev3-L979F mutation had little to no effect on mutagenesis in an ogg1Delta background, which cannot repair 8-oxo-guanine in DNA. UV-induced can1 mutants from rev3-L979F and rad30Deltarev3-L979F strains primarily contained base substitutions and complex mutations, suggesting error-prone bypass of UV photoproducts by L979F pol zeta. Spontaneous mutation rates in rev3-L979F and rev3-L979M strains are elevated by about two-fold overall and by two- to eight-fold for C to G transversions and complex mutations, both of which are known to be generated by wild-type pol zetain vitro. These results indicate that Rev3p-Leu979 replacements reduce the fidelity of DNA synthesis by yeast pol zetain vivo. In conjunction with earlier studies, the data establish that the conserved amino acid at the active site location occupied by Leu979 is critical for the fidelity of all four yeast B family polymerases. Reduced fidelity with retention of robust polymerase activity suggests that the homologous rev3-L979F allele may be useful for analyzing pol zeta functions in mammals, where REV3 deletion is lethal.     

Specificity of the Yeast Rev3Δ Antimutator and Rev3 Dependency of the Mutator Resulting from a Defect (Rad1Δ) 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 (rad1Δ). 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 rev3Δ-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. rev3Δ also greatly diminished the magnitude of the rad1Δ mutator, but not to the rev3Δ antimutator level, implicating REV3-dependent and independent processes in the rad1Δ mutator effect. However, the specificity of the rev3Δ antimutator suggested that the same REV3-dependent processes gave rise to the majority of spontaneous mutations in the RAD1 and rad1Δ strains.     

The Saccharomyces Cerevisiae Rad30 Gene, a Homologue of Escherichia Coli Dinb and Umuc, Is DNA Damage Inducible and Functions in a Novel Error-Free Postreplication Repair Mechanism

Damage-inducible mutagenesis in prokaryotes is largely dependent upon the activity of the UmuD'C-like proteins. Since many DNA repair processes are structurally and/or functionally conserved between prokaryotes and eukaryotes, we investigated the role of RAD30, a previously uncharacterized Saccharomyces cerevisiae DNA repair gene related to the Escherichia coli dinB, umuC and S. cerevisiae REV1 genes, in UV resistance and UV-induced mutagenesis. Similar to its prokaryotic homologues, RAD30 was found to be damage inducible. Like many S. cerevisiae genes involved in error-prone DNA repair, epistasis analysis clearly places RAD30 in the RAD6 group and rad30 mutants display moderate UV sensitivity reminiscent of rev mutants. However, unlike rev mutants, no defect in UV-induced reversion was seen in rad30 strains. While rad6 and rad18 are both epistatic to rad30, no epistasis was observed with rev1, rev3, rev7 or rad5, all of which are members of the RAD6 epistasis group. These findings suggest that RAD30 participates in a novel error-free repair pathway dependent on RAD6 and RAD18, but independent of REV1, REV3, REV7 and RAD5.     

UV and chemical mutagenesis in rev7 mutants of yeast

Summary We have examined induced mutagenesis in rev7-1 mutants of Baker's yeast' Saccharomyces cerevisiae, using a variety of contrasting test systems and several different mutagens. UV-induced reversion frequencies of the ochre allele arg4-17, the putative missense allele ilv1-92 and the frameshift allele his4-38 were 10 to 200 fold lower in haploid and diploid rev7-1 mutants compared with wild type strains, but UV-induced reversion frequencies of the frameshift allele leu2-3 and the proline missense allele cyc1-115 were reduced only a few fold. Ilv1-92 reversion frequencies induced by methyl methane sulfonate or by N-methyl-N-nitro-N-nitrosoguanidine were 10 to 20 times lower in rev7-1 mutants, but normal frequencies of these revertants were induced with ethyl methane sulfonate, even though rev7-1 strains are slightly sensitive to this mutagen as well as to the others tested. We conclude that the rev7 mutants, like the rev3 mutants they closely resemble, have a substantial but not total deficiency concerning induced mutagenesis.     

Mutational Specificity of Ultraviolet Light in ESCHERICHIA COLI with and without the R Plasmid Pkm101

Plasmid pKM101 provides UV protection and increases the frequency of spontaneous and UV-induced mutations in Escherichia coli. By analyzing reversion patterns of defined trpA alleles, we showed that pKM101 altered the mutational specificity of UV-induced mutations. Certain UV-induced base-pair substitutions were strongly enhanced, while others were decreased in frequency in the presence of pKM101. This result suggests an interaction between cellular misrepair and an error-prone repair function(s) provided by pKM101. We have also examined UV mutational specificity in the absence of pKM101 and found the following: (1) UV preferentially enhances missense, as well as nonsense, intergenic suppressor mutations; (2) UV causes all possible base-pair substitutions as well as frameshift mutations; (3) G·C base pairs are more susceptible to UV mutagenesis than A·T base pairs at the same nucleotide positions; and (4) UV-induced mutations can occur at nucleotide positions that are not part of pyrimidine-pyrimidine sequences.     

REV3 is required for spontaneous but not methylation damage-induced mutagenesis of Saccharomyces cerevisiae cells lacking O6-methylguanine DNA methyltransferase

O6-methylguanine (O6-MeG) DNA methyltransferase (MTase) removes the methyl group from a DNA lesion and directly restores DNA structure. It has been shown previously that bacterial and yeast cells lacking such MTase activity are not only sensitive to killing and mutagenesis by DNA methylating agents, but also exhibit an increased spontaneous mutation rate. In order to understand molecular mechanisms of endogenous DNA alkylation damage and its effects on mutagenesis, we determined the spontaneous mutational spectra of the SUP4-o gene in various Saccharomyces cerevisiae strains. To our surprise, the mgt1 mutant deficient in DNA repair MTase activity exhibited a significant increase in G:C-->C:G transversions instead of the expected G:C-->A:T transition. Its mutational distribution strongly resembles that of the rad52 mutant defective in DNA recombinational repair. The rad52 mutational spectrum has been shown to be dependent on a mutagenesis pathway mediated by REV3. We demonstrate here that the mgt1 mutational spectrum is also REV3-dependent and that the rev3 deletion offsets the increase of the spontaneous mutation rate seen in the mgt1 strains. These results indicate that the eukaryotic mutagenesis pathway is directly involved in cellular processing of endogenous DNA alkylation damage possibly by the translesion bypass of lesions at the cost of G:C-->C:G transversion mutations. However, the rev3 deletion does not affect methylation damage-induced killing and mutagenesis of the mgt1 mutant, suggesting that endogenous alkyl lesions may be different from O6-MeG.     

UV-induced reversion of his4 frameshift mutations in rad6, rev1, and rev3 mutants of yeast

Summary The UV-induced reversion of two his4 frameshift alleles was much reduced in rad6 mutants of Saccharomyces cerevisiae, an observation that is consistent with the hypothesis that RAD6 function is required for the induction of all types of genetic alteration in misrepair mutagenesis. The reversion of these his4 alleles, together with two others of the same type, was also reduced in rev1 and rev3 mutant strains; in these, however, the extent of the reduction varied considerably with test allele used, in a manner analogous to the results in these strains for base repair substitution test alleles. The general features of UV-induced frameshift and substitution mutagenesis therefore appear quite similar, indicating that they may depend on related processes. If this conclusion is correct, greater attention must be given to integrating models which account for the production of nucleotide additions and deletions into those concerning misrepair mutagenesis.     

A Role for Rev3 in Mutagenesis during Double-Strand Break Repair in Saccharomyces Cerevisiae

Recombinational repair of double-strand breaks (DSBs), traditionally believed to be an error-free DNA repair pathway, was recently shown to increase the frequency of mutations in a nearby interval. The reversion rate of trp1 alleles (either nonsense or frameshift mutations) near an HO-endonuclease cleavage site is increased at least 100-fold among cells that have experienced an HO-mediated DSB. We report here that in strains deleted for rev3 this DSB-associated reversion of a nonsense mutation was greatly decreased. Thus REV3, which encodes a subunit of the translesion DNA polymerase &, was responsible for the majority of these base substitution errors near a DSB. However, rev3 strains showed no decrease in HO-stimulated recombination, implying that another DNA polymerase also functioned in recombinational repair of a DSB. Reversion of trp1 frameshift alleles near a DSB was not reduced in rev3 strains, indicating that another polymerase could act during DSB repair to make these frameshift errors. Analysis of spontaneous reversion in haploid strains suggested that Rev3p had a greater role in making point mutations than in frameshift mutations.     

Effect of Genes Controlling Radiation Sensitivity on Chemically Induced Mutations in SACCHAROMYCES CEREVISIAE

The effect of 16 different genes (rad) conferring radiation sensitivity on chemically induced reversion in the yeast Saccharomyces cerevisiae was determined. The site of reversion used was a well-defined chain initiation mutant mapping in the structural gene coding for iso-1-cytochrome c. High doses of EMS and HNO₂ resulted in decreased reversion of cyc1–131 in rad6, rad9 and rad15 strains compared to the normal RAD+ strains. In addition, rad52 greatly decreased EMS reversion of cyc1–131 but had not effect on HNO ₂-induced reversion; rad18, on the other hand, increased HNO ₂-induced reversion but did not alter EMS-induced reversion. When NQO was used as the mutagen, every rad gene tested, except for rad14 , had an effect on reversion; rad6, rad9, rad15, rad17, rad18, rad22, rev1, rev2 and rev3 lowered NQO reversion while rad1, rad2, rad3, rad4, rad10, rad12 and rad16 increased it compared to the RAD+ strain. The effect of rad genes on chemical mutagenesis is discussed in terms of their effect on UV mutagenesis. It is concluded that although the nature of the repair pathways may differ for UV- and chemically-induced mutations in yeast, a functional repair system is required for the induction of mutation by the chemical agents NQO, EMS and HNO₂.     

Analysis of mutagenic DNA repair in a thermoconditional repair mutant of Saccharomyces cerevisiae. II. Influence of cycloheximide on UV-irradiated exponentially growing rev2ts cells

The time course of REV2 dependent recovery from prelethal UV damage and UV-induced locus-specific reversion of the his5-2 allele was determined in temperature-shift experiments by use of a thermoconditional allele of the rev2 gene (rad5-8, rev2ts). In UV-irradiated, exponentially growing rev2ts cells the REV2 dependent repair activity persists for up to 8 h at permissive temperature (23 degrees C), while the REV2 dependent mutagenic process is mostly completed within 2 h. The REV2 dependent process in exponentially growing cells is highly impaired by inhibition of protein synthesis. However, a REV2 dependent repair activity independent of de novo synthesis is detectable, even in the presence of up to 200 micrograms/ml cycloheximide, a response not found in stationary phase cells. Thus, the REV2 dependent process seems to be partially constitutive in exponentially growing cells. Additionally, exponentially growing rev2ts cells were considerably more UV-sensitive at restrictive temperature (36 degrees C) than were stationary phase cells.     

REV3, a Saccharomyces cerevisiae gene whose function is required for induced mutagenesis, is predicted to encode a nonessential DNA polymerase

We have cloned the REV3 gene of Saccharomyces cerevisiae by complementation of the rev3 defect in UV-induced mutagenesis. The nucleotide sequence of this gene encodes a predicted protein of Mr 172,956 showing significant sequence similarity to Epstein-Barr virus DNA polymerase and to other members of a class of DNA polymerases including human DNA polymerase alpha and yeast DNA polymerase I. REV3 protein shows less sequence identity, and presumably a more distant evolutionary relationship, to the latter two enzymes than they do to each other. Haploids carrying a complete deletion of REV3 are viable. We suggest that induced mutagenesis in S. cerevisiae depends on a specialized DNA polymerase that is not required for other replicative processes. REV3 is located 2.8 centimorgans from CDC60, on chromosome XVI.     

The isolation and characterization of ngm2, a mutation that affects nitrosoguanidine mutagenesis in yeast

Summary We have isolated and characterized a new mutant of Saccharomyces cerevisiae, carrying a single mutant allele that we designate ngm2-1, which is defective with respect to induced mutagenesis. This mutant was isolated by screening mutagenized clones for reduced frequencies of reversion of the his1-7 allele, induced by N-methyl-N-nitro-N-nitrosoguanidine. As judged by the reversion of his1-7 and ilv1-92, ngm2-1 mutant strains are also deficient with respect to mutability induced by methyl methane sulfonate, ethyl methane sulfonate and, at least partially, by UV. UV-induced reversion of the ochre mutation arg4-17 and the frameshift mutation his4-38 was not much affected by ngm2-1, however. Like rev3 and rev7 mutations, ngm2-1 also has little influence on the reversion of the proline missense allele, cyc1-115. Ngm2-1 mutants are only at best very slightly more sensitive to the toxicity of the four mutagens used, and homozygous diploids sporulate normally.     

HSM2 (HMO1) gene participates in mutagenesis control in yeast Saccharomyces cerevisiae

We have previously reported about a new Saccharomyces cerevisiae mutation, hsm2-1, that results in increase of both spontaneous and UV-induced mutation frequencies but does not alter UV-sensitivity. Now HSM2 gene has been genetically and physically mapped and identified as a gene previously characterized as HMO1, a yeast homologue of human high mobility group genes HMG1/2. We found that hsm2 mutant is slightly deficient in plasmid-borne mismatch repair. We tested UV-induced mutagenesis in double mutants carrying hsm2-1 mutation and a mutation in a gene of principal damaged DNA repair pathways (rad2 and rev3) or in a mismatch repair gene (pms1 and recently characterized in our laboratory hsm3). The frequency of UV-induced mutations in hsm2 rev3 was not altered in comparison with single rev3 mutant. In contrast, the interaction of hsm2-1 with rad2 and pms1 was characterized by an increased frequency of UV-induced mutations in comparison with single rad2 and pms1 mutants. The UV-induced mutation frequency in double hsm2 hsm3 mutant was lower than in the single hsm2 and hsm3 mutants. The role of the HSM2 gene product in control of mutagenesis is discussed.     

Error-free RAD52 pathway and error-prone REV3 pathway determines spontaneous mutagenesis in Saccharomyces cerevisiae

Using the CAN1 gene in haploid cells or heterozygous diploid cells, we characterized the effects of mutations in the RAD52 and REV3 genes of Saccharomyces cerevisiae in spontaneous mutagenesis. The mutation rate was 5-fold higher in the haploid rad52 strain and 2.5-fold lower in rev3 than in the wild-type strain. The rate in the rad52 rev3 strain was as low as in the wild-type strain, indicating the rad52 mutator phenotype to be dependent on REV3. Sequencing indicated that G:C-->T:A and G:C-->C:G transversions increased in the rad52 strain and decreased in the rev3 and rad52 rev3 strains, suggesting a role for REV3 in transversion mutagenesis. In diploid rev3 cells, frequencies of can1Delta::LEU2/can1Delta::LEU2 from CAN1/can1Delta::LEU2 due to recombination were increased over the wild-type level. Overall, in the absence of RAD52, REV3-dependent base-substitutions increased, while in the absence of REV3, RAD52-dependent recombination events increased. We further found that the rad52 mutant had an increased rate of chromosome loss and the rad52 rev3 double mutant had an enhanced chromosome loss mutator phenotype. Taken together, our study indicates that the error-free RAD52 pathway and error-prone REV3 pathway for rescuing replication fork arrest determine spontaneous mutagenesis, recombination, and genome instability.     

The Saccharomyces cerevisiae rev6-1 mutation, which inhibits both the lesion bypass and the recombination mode of DNA damage tolerance, is an allele of POL30, encoding proliferating cell nuclear antigen

The rev6-1 allele was isolated in a screen for mutants deficient for UV-induced reversion of the frameshift mutation his4-38. Preliminary testing showed that the rev6-1 mutant was substantially deficient for UV-induced reversion of arg4-17 and ilv1-92 and markedly UV sensitive. Unlike other REV genes, which encode DNA polymerases and an associated subunit, REV6 has been found to be identical to POL30, which encodes proliferating cell nuclear antigen (PCNA), the subunit of the homotrimeric sliding clamp, in which the rev6-1 mutation produces a G178S substitution. This substitution appears to abolish all DNA damage-tolerance activities normally carried out by the RAD6/RAD18 pathway, including translesion replication by DNA polymerase zeta/Rev1 and DNA polymerase eta, and the error-free, recombination-dependent component of this pathway, but has little effect on the growth rate, suggesting that G178S may prevent ubiquitination of lysine 164 in PCNA. We also find that rev6-1 mutation can be fully complemented by a centromere-containing, low copy-number plasmid carrying POL30, despite the presumed occurrence in the mutant of sliding clamp assemblies that contain between one and three G178S PCNA monomers as well as the fully wild-type species.     

The in vivo characterization of translesion synthesis across UV-induced lesions in Saccharomyces cerevisiae: insights into Pol zeta- and Pol eta-dependent frameshift mutagenesis

UV irradiation, a known carcinogen, induces the formation of dipyrimidine dimers with the predominant lesions being cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone adducts (6-4PPs). The relative roles of the yeast translesion synthesis DNA polymerases Pol zeta and Pol eta in UV survival and mutagenesis were examined using strains deficient in one or both polymerases. In addition, photoreactivation was used to specifically remove CPDs, thus allowing an estimate to be made of the relative contributions of CPDs vs. 6-4PPs to overall survival and mutagenesis. In terms of UV-induced mutagenesis, we focused on the +1 frameshift mutations detected by reversion of the lys2deltaA746 allele, as Pol zeta produces a distinct mutational signature in this assay. Results suggest that CPDs are responsible for most of the UV-associated toxicity as well as for the majority of UV-induced frameshift mutations in yeast. Although the presence of Pol eta generally suppresses UV-induced mutagenesis, our data suggest a role for this polymerase in generating some classes of +1 frameshifts. Finally, the examination of frameshift reversion spectra indicates a hierarchy between Pol eta and Pol zeta with respect to the bypass of UV-induced lesions.     

Isolation and Characterization of Amber Suppressors in Yeast

Nonsense suppressors were obtained in a haploid yeast strain containing eight nutritional mutations, that are assumed to be amber or ochre, and the cyc1-179 amber mutation that has a UAG codon corresponding to position 9 in iso-1-cytochrome c. Previous studies established that the biosynthesis and function of iso-1-cytochrome c is compatible with replacements at position 9 of amino acids having widely different structures (Stewart and Sherman 1972). UV-induced revertants, selected on media requiring the reversion of one or two of the amber nutritional markers, were presumed to contain a suppressor if there was the unselected reversion of at least one other marker. The 1088 suppressors that were isolated could be divided into 78 phenotypic classes. Only 43 suppressors of three classes caused the production of more than 50% of the normal amount of iso-1-cytochrome c in the cyc1-179 strain. Genetic analyses indicated that all of these highly efficient amber suppressors are allelic to one or another of the eight suppressors which cause the insertion of tyrosine at ochre (UAA) codons (Gilmore, Stewart and Sherman 1971). Furthermore, only tyrosine has been identified at position 9 in iso-1-cytochrome c in cyc1-179 strains suppressed with these efficient amber suppressors.