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.     

Spontaneous mutability in UV-sensitive excision-defective strains of Saccharomyces.

The genes RAD1, RAD2, RAD3 and RAD4 encode enzymes in the pathway leading to excision repair of UV-induced DNA damage in Saccharomyces cerevisiae. Four mutant alleles of these loci (rad1-1, rad2-2, rad3-12, and rad4-3) were studied for their effect on spontaneous reversion rate to lysine and histidine independence, by means of the 1000-compartment fluctuation test of von Borstel, Cain and Steinberg. Of these four excision-defective alleles, only rad3-12 was found to substantially increase the spontaneous reversion rate of the nonsense-suppressible lys1-1 allele, both through locus reversion as well as by forward mutation at one of eight suppressor loci. Similarly, only rad3-12 conferred a considerable increase in the reversion frequency of the missense his1-7 mutant. As the RAD3 gene product is believed to mediate the first step in the excision-repair pathway, it is assumed that spontaneous lesions in the rad3 strain are channelled into a mutagenic repair pathway, thus accounting for the enhanced spontaneous mutation rate.     

The Saccharomyces cerevisiae RAD9, RAD17, RAD24 and MEC3 genes are required for tolerating irreparable,ultraviolet-induced DNA damage.

In wild-type Saccharomyces cerevisiae, a checkpoint slows the rate of progression of an ongoing S phase in response to exposure to a DNA-alkylating agent. Mutations that eliminate S phase regulation also confer sensitivity to alkylating agents, leading us to suggest that, by regulating the S phase rate, cells are either better able to repair or better able to replicate damaged DNA. In this study, we determine the effects of mutations that impair S phase regulation on the ability of excision repair-defective cells to replicate irreparably UV-damaged DNA. We assay survival after UV irradiation, as well as the genetic consequences of replicating a damaged template, namely mutation and sister chromatid exchange induction. We find that RAD9, RAD17, RAD24, and MEC3 are required for UV-induced (although not spontaneous) mutagenesis, and that RAD9 and RAD17 (but not REV3, RAD24, and MEC3) are required for maximal induction of replication-dependent sister chromatid exchange. Therefore, checkpoint genes not only control cell cycle progression in response to damage, but also play a role in accommodating DNA damage during replication.     

Radiosensitive and mitotic recombination phenotypes of the Saccharomyces cerevisiae dun1 mutant defective in DNA damage-inducible gene expression.

The biological significance of DNA damage-induced gene expression in conferring resistance to DNA-damaging agents is unclear. We investigated the role of DUN1-mediated, DNA damage-inducible gene expression in conferring radiation resistance in Saccharomyces cerevisiae. The DUN1 gene was assigned to the RAD3 epistasis group by quantitating the radiation sensitivities of dun1, rad52, rad1, rad9, rad18 single and double mutants, and of the dun1 rad9 rad52 triple mutant. The dun1 and rad52 single mutants were similar in terms of UV sensitivities; however, the dun1 rad52 double mutant exhibited a synergistic decrease in UV resistance. Both spontaneous intrachromosomal and heteroallelic gene conversion events between two ade2 alleles were enhanced in dun1 mutants, compared to DUN1 strains, and elevated recombination was dependent on RAD52 but not RAD1 gene function. Spontaneous sister chromatid exchange (SCE), as monitored between truncated his3 fragments, was not enhanced in dun1 mutants, but UV-induced SCE and heteroallelic recombination were enhanced. Ionizing radiation and methyl methanesulfonate (MMS)-induced DNA damage did not exhibit greater recombinogenicity in the dun1 mutant compared to the DUN1 strain. We suggest that one function of DUN1-mediated DNA damage-induced gene expression is to channel the repair of UV damage into a nonrecombinogenic repair pathway.     

A role for histone H2B during repair of UV-induced DNA damage in Saccharomyces cerevisiae

To investigate the role of the nucleosome during repair of DNA damage in yeast, we screened for histone H2B mutants that were sensitive to UV irradiation. We have isolated a new mutant, htb1-3, that shows preferential sensitivity to UV-C. There is no detectable difference in bulk chromatin structure or in the number of UV-induced cis-syn cyclobutane pyrimidine dimers (CPD) between HTB1 and htb1-3 strains. These results suggest a specific effect of this histone H2B mutation in UV-induced DNA repair processes rather than a global effect on chromatin structure. We analyzed the UV sensitivity of double mutants that contained the htb1-3 mutation and mutations in genes from each of the three epistasis groups of RAD genes. The htb1-3 mutation enhanced UV-induced cell killing in rad1Delta and rad52Delta mutants but not in rad6Delta or rad18Delta mutants, which are defective in postreplicational DNA repair (PRR). When combined with other mutations that affect PRR, the histone mutation increased the UV sensitivity of strains with defects in either the error-prone (rev1Delta) or error-free (rad30Delta) branches of PRR, but did not enhance the UV sensitivity of a strain with a rad5Delta mutation. When combined with a ubc13Delta mutation, which is also epistatic with rad5Delta, the htb1-3 mutation enhanced UV-induced cell killing. These results suggest that histone H2B acts in a novel RAD5-dependent branch of PRR.     

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.     

Frameshift mutagenesis by ultraviolet and gamma radiations in Salmonella: SOS inducibility and effects of DNA polymerase I

Ultraviolet (UV) and gamma-induced mutagenesis have been studied using a doubly auxotrophic strain of Salmonella typhimurium carrying the amber leuA150 mutation (which reverts by base-pair substitution) and the frameshift hisC3076 marker (which reverts by compensating frameshifts). In the initially constructed LT2 background, both markers were poorly revertible by UV and essentially non-revertible by gamma-radiation. A derivative of this strain carrying the mutation-enhancing plasmid pKM101 was however readily reverted by both UV and gamma, with either Leu+ (base substitution) or His+ (frameshift) revertants being observed on appropriate selective media. Photoreactivation experiments suggested that the lesions leading to formation of the two types of mutagenic event were similar if not identical. Support for this suggestion was obtained when it was found that yields of both types of UV-induced revertant were significantly increased in an excision-deficient background, while no revertants of either type were found in a recA background. Yields of gamma-induced revertants were not greatly altered in a uvrB background, but were also reduced to zero (for both markers) in the recA background. These results are consistent with what has previously been well-documented for UV and gamma-induced base-pair substitution mutagenesis, and serve to emphasize the similarities between base-pair substitution mutagenesis and frameshift mutagenesis by these agents. There are differences, however, since although UV-induced reversion of the leuA150 marker was little affected and gamma-induced reversion of leuA150 was somewhat reduced in the presence of a polA mutation (polA3), the yields of His+ frameshift revertants were significantly increased in the polA3 background following treatment with either UV or gamma. Thus while inducible DNA repair (SOS repair) appears to be involved in generating both types of mutational event following either UV- or gamma-irradiation, at some stage in the processing of premutational lesions the level (or type) of DNA polymerase I activity in the cell seems to have an important role in determining whether or not frameshifts or base-pair substitutions will be produced at a particular frequency.     

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.     

REV7, a new gene concerned with UV mutagenesis in yeast

Summary Three allelic mutations of a new yeast gene, which we have named REV7, have been isolated by testing 313 methyl methane sulfonate sensitive mutants for UV-induced reversion of a lys2 allele. Rev7 mutants are markedly deficient with respect to UV-induced reversion of lys2, are slightly sensitive to UV and appear to be in the RAD6 epistasis group for UV survival. Rev7-1, which is probably an amber mutation, does not appear to affect sporulation in homozygous diploids. The REV7 gene is located about 12 cM distal to HIS5 on chromosome IX.     

Evidence for excision repair in promitochondrial DNA of anaerobic cells of Saccharomyces cerevisiae.

The respiratory adaptation (i.e., essentially mitochondrial biogenesis) in the excision repair-defective rad3-type mutants of Saccharomyces cerevisiae undergoing transition from the anaerobic to the aerobic state is found to be far more sensitive to 254-nm ultraviolet radiation (UV) than that of the RAD wild-type strain. We confirm that mitochondria of aerobic cells of a RAD strain lack the excision repair capacity of UV-induced pyrimidine dimers at all doses tested (1-15 J/m2). In contrast, in promitochondria of anaerobic cells of the wild-type strain excision repair appears to take place. This process is very efficient at low doses (at 0.5-5 J/m2 100% of the UV endonuclease-sensitive sites disappear), whereas at high doses its efficiency is reduced by about 50%. The promitochondrial excision repair of pyrimidine dimers appears to be under nuclear control since it is blocked in the rad2 mutant. Finally photoreactivation is found to be operating in nuclei, mitochondria and promitochondria.     

Mutagenesis in cloned yeast genes. The effect of mutation rad2 on the frequency of gene mutation in plasmid and chromosome

The influence of rad2 mutation blocking incision of pyrimidine dimers on frequency of UV-light and 6-hydroxylaminopurine (6-GAP)-induced adenine-independent revertants was studied in the strains of Saccharomyces cerevisiae containing the same mutant allele of gene ADE2 in episomic plasmid and in chromosome. It was shown that the strains carrying the ade2 mutation in chromosome and in plasmid did not differ in sensitivity to lethal action of UV-light and 6-GAP. However, in the plasmid rad2 strain reversions were induced by UV-light more frequently (approximately 100 times), as compared to the chromosome strain. We observed no significant differences between reversion frequencies in plasmid and chromosome RAD strains. The tendency to enhanced 6-GAP-induced mutagenesis, less sharply expressed, was observed in the chromosome rad2 strain, as compared to the plasmid one. However, the plasmid RAD strain was characteristic of higher reversion frequency induced by 6-GAP, as compared to the chromosome strain. The possible mechanisms of these phenomena are discussed.     

The Srs2 Suppressor of Rad6 Mutations of Saccharomyces Cerevisiae Acts by Channeling DNA Lesions into the Rad52 DNA Repair Pathway

rad6 mutants of Saccharomyces cerevisiae are defective in the repair of damaged DNA, DNA damage induced mutagenesis, and sporulation. In order to identify genes that can substitute for RAD6 function, we have isolated genomic suppressors of the UV sensitivity of rad6 deletion (rad6 delta) mutations and show that they also suppress the gamma-ray sensitivity but not the UV mutagenesis or sporulation defects of rad6. The suppressors show semidominance for suppression of UV sensitivity and dominance for suppression of gamma-ray sensitivity. The six suppressor mutations we isolated are all alleles of the same locus and are also allelic to a previously described suppressor of the rad6-1 nonsense mutation, SRS2. We show that suppression of rad6 delta is dependent on the RAD52 recombinational repair pathway since suppression is not observed in the rad6 delta SRS2 strain containing an additional mutation in either the RAD51, RAD52, RAD54, RAD55 or RAD57 genes. Possible mechanisms by which SRS2 may channel unrepaired DNA lesions into the RAD52 DNA repair pathway are discussed.     

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.     

Effect of SOS-induced Pol II, Pol IV, and Pol V DNA polymerases on UV-induced mutagenesis and MFD repair in Escherichia coli cells

Irradiation of organisms with UV light produces genotoxic and mutagenic lesions in DNA. Replication through these lesions (translesion DNA synthesis, TSL) in Escherichia coli requires polymerase V (Pol V) and polymerase III (Pol III) holoenzyme. However, some evidence indicates that in the absence of Pol V, and with Pol III inactivated in its proofreading activity by the mutD5 mutation, efficient TSL takes place. The aim of this work was to estimate the involvement of SOS-inducible DNA polymerases, Pol II, Pol IV and Pol V, in UV mutagenesis and in mutation frequency decline (MFD), a mechanism of repair of UV-induced damage to DNA under conditions of arrested protein synthesis. Using the argE3-->Arg(+) reversion to prototrophy system in E. coli AB1157, we found that the umuDC-encoded Pol V is the only SOS-inducible polymerase required for UV mutagenesis, since in its absence the level of Arg(+) revertants is extremely low and independent of Pol II and/or Pol IV. The low level of UV-induced Arg(+) revertants observed in the AB1157mutD5DumuDC strain indicates that under conditions of disturbed proofreading activity of Pol III and lack of Pol V, UV-induced lesions are bypassed without inducing mutations. The presented results also indicate that Pol V may provide substrates for MFD repair; moreover, we suggest that only those DNA lesions which result from umuDC-directed UV mutagenesis are subject to MFD repair.     

Inducible error-prone repair in yeast. Suppression by heat shock

The production of reversion mutations in wild-type, diploid Saccharomyces cerevisiae by the alkylating agents N-methyl-N'-nitro- N-nitrosoguanidine (MNNG) and methylnitrosourea (MNU) was suppressed in cells previously treated with a heat shock, or the protein synthesis inhibitor, cycloheximide. The same cells previously treated with a heat shock, or the protein synthesis inhibitor, cycloheximide. The same treatment after mutagen exposure did not lower the induced mutation frequency. In split-dose experiments, a first MNNG exposure prevented subsequent heat (or cycloheximide) treatment from blocking mutation by a second, later mutagen exposure. These data suggest that, in yeast, MNNG or MNU induces an error-prone DNA-repair system, and that this induction is blocked by protein-synthesis inhibitors. The specificity of this system for different types of DNA damage was investigated using a variety of other mutagenic agents. A prior heat shock did not suppress mutation produced by exposure to ethyl methanesulfonate, ethylnitrosourea, 8-methoxypsoralen + UVA, or gamma-radiation. Partial suppression was observed in cells exposed to methyl methanesulfonate or to 254-nm ultraviolet light. These results indicate that, unlike the SOS system of E. coli, this inducible error-prone process of yeast is responsive to only certain mutagens. Heat shock suppression of mutation produced by MNNG exposure was also demonstrated in wild-type haploid cells, as well as haploid strains mutant in representative genes of the RAD52 epistasis group (rad52, rad53, rad54), the RAD3 epistasis group (rad1, rad2, rad3) and the RAD6 epistasis group (rad9, rad18). The rad6 mutant itself was immutable with MNNG and therefore untestable by these techniques. These data indicate that this error-prone repair system is not absolutely dependent on the integrity of the RAD52 (recombination) or the RAD3 (excision) systems, or on at least some parts of the RAD6 system.     

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.     

a/alpha-specific effect on the mms3 mutation on ultraviolet mutagenesis in Saccharomyces cerevisiae.

A new gene involved in error-prone repair of ultraviolet (UV) damage has been identified in Saccharomyces cerevisiae by the mms3-1 mutation. UV-induced reversion is reduced in diploids that are homozygous for mms3-1, only if they are also heterozygous (MATa/MAT alpha) at the mating type locus. The mms3-1 mutation has no effect on UV-induced reversion either in haploids or MATa/MATa or MAT alpha/MAT alpha diploids. The mutation confers sensitivity to UV and methyl methane sulfonate in both haploids and diploids. Even though mutation induction by UV is restored to wild-type levels in MATa/MATa mms3-1/mms3-1 or MAT alpha/MAT alpha mms3-1/mms3-1 diploids, such strains still retain sensitivity to the lethal effects of UV. Survival after UV irradiation in mms3-1 rad double mutant combinations indicates that mms3-1 is epistatic to rad6-1 whereas non-epistatic interactions are observed with rad3 and rad52 mutants. When present in the homozygous state in MATa/MAT alpha his1-1/his1-315 heteroallelic diploids, mms3-1 was found to lower UV-induced mitotic recombination.