Requirement of HSM3 gene for spontaneous mutagenesis in Saccharomyces cerevisiae.

In this work, we studied the influence of hsm3 mutation on spontaneous mutagenesis in actively and slowly dividing cells. We demonstrated that the spontaneous mutation rates in the hsm3 mutant and the wild type strain were similar in actively dividing cells. However, during 15-day cultivation of both strains we observed higher accumulation of mutants in the hsm3 strain compared with those in the wild type cells. Effect of accumulation of spontaneous mutants was observed in slowly dividing cells in the rad1, rad2, rad14, rad54, and pms1, but it was absent in the rev3, pol2 and pol3 mutants. Combinations of the hsm3 mutation with the pol3-01, pol2-04 and pms1Delta mutations decreased significantly the level of spontaneous mutagenesis in rapidly growing cells. The hsm3 mutation suppressed synthetic lethality in the hsm3 pol3-01 pms1 triple mutant and dramatically increased the spontaneous mutation rate in comparison with double mutant. The introduction of the hsm3 mutation in NER-mutants led to considerably increasing of the spontaneous mutation level. The double hsm3 rev3, hsm3 rad54 and hsm3 pms1Delta mutants showed lower spontaneous mutation rate compared with the single mutants in rapidly dividing cells. The combination the hsm3 mutation with all studied mutations characterized by different degree of increase of spontaneous mutagenesis in slowly dividing cells. The participation of the Hsm3p in spontaneous mutagenesis in slowly and activity dividing yeast cells is discussed.

The yeast HSM3 gene acts in one of the mismatch repair pathways.

Mutants with enhanced spontaneous mutability (hsm) to canavanine resistance were induced by N-methyl-N-nitrosourea in Saccharomyces cerevisiae. One bearing the hsm3-1 mutation was used for this study. This mutation does not increase sensitivity to the lethal action of different mutagens. The hsm3-1 mutation produces a mutator phenotype, enhancing the rates of spontaneous mutation to canavanine resistance and reversions of lys1-1 and his1-7. This mutation increases the rate of intragenic mitotic recombination at the ADE2 gene. The ability of the hsm3 mutant to correct DNA heteroduplex is reduced in comparison with the wild-type strain. All these phenotypes are similar to ones caused by pms1, mlhl and msh2 mutations. In contrast to these mutations, hsm3-1 increases the frequency of ade mutations induced by 6-HAP and UV light. Epistasis analysis of double mutants shows that the PMS1 and HSM3 genes control different mismatch repair systems. The HSM3 gene maps to the right arm of chromosome II, 25 cM distal to the HIS7 gene. Strains that bear a deleted open reading frame YBR272c have the genetic properties of the hsm3 mutant. The HSM3 product shows weak similarity to predicted products of the yeast MSH genes (homologs of the Escherichia coli mutS gene). The HSM3 gene may be a member of the yeast MutS homolog family, but its function in DNA metabolism differs from the functions of other yeast MutS homologs.

Saccharomyces cerevisiae mutants with enhanced induced mutation and altered mitotic gene conversion.

We have developed a method to isolate yeast (Saccharomyces cerevisiae) mutants with enhanced induced mutagenesis based on nitrous acid-induced reversion of the ade2-42 allele. Six mutants have been isolated and designated him (high induced mutagenesis), and 4 of them were studied in more detail. The him mutants displayed enhanced reversion of the ade2-42 allele, either spontaneous or induced by nitrous acid, UV light, and the base analog 6-N-hydroxylaminopurine, but not by gamma-irradiation. It is worth noting that the him mutants turned out not to be sensitive to the lethal effects of the mutagens used. The enhancement in mutation induced by nitrous acid, UV light, and 6-N-hydroxylaminopurine has been confirmed in a forward-mutation assay (induction of mutations in the ADE1, ADE2 genes). The latter agent revealed the most apparent differences between the him mutants and the wild-type strain and was, therefore, chosen for the genetic analysis of mutants, him mutations analyzed behaved as a single Mendelian trait; complementation tests indicated 3 complementation groups (HIM1, HIM2, and HIM3), each containing 1 mutant allele. Uracil-DNA glycosylase activity was determined in crude cell extracts, and no significant differences between the wild-type and him strains were detected. Spontaneous mitotic gene conversion at the ADE2 locus is altered in him1 strains, either increased or decreased, depending on the particular heteroallelic combination. Genetic evidence strongly suggests him mutations to be involved in a process of mismatch correction of molecular heteroduplexes.

The rad2 mutation affects the molecular nature of UV and acridine-mustard-induced mutations in the ADE2 gene of Saccharomyces cerevisiae.

We have studied the molecular nature of ade2 mutations induced by UV light and bifunctional acridine-mustard (BAM) in wild-type (RAD) and in excision-deficient (rad2) strains of the yeast, Saccharomyces cerevisiae. In the RAD strain, UV causes 45% GC----AT transitions among all mutations; in the rad2 strain this value is 77%. BAM was shown to be highly specific for frameshift mutagenesis: 60% frameshifts in the RAD strain, and as many as 84% frameshifts in the rad2 strain were induced. Therefore, the rad2 mutation affects the specificity of UV- and BAM-induced mutagenesis in yeast. Experimental data agree with the view that the majority of mutations in the RAD strain are induced by a prereplicative mechanism, whereas mutations in the RAD strain are induced by a prereplicative mechanism, whereas mutations in the rad2 strain are predominantly postreplicative events. Our results also suggest that: cytosine-containing photoproducts are the substances responsible for major premutational damage to cytosine-containing photoproducts are the substances responsible for major premutational damage to DNA; a fraction of the mutations may arise in the course of excision repair of UV photoproducts.