The effect of him mutations characterized by enhanced induced mutagenesis on the spontaneous mitotic recombination in the ADE2 gene in Saccharomyces cerevisiae

We have studied the influence of him1, him2, him3 and himX mutations on the frequency of spontaneous mitotic gene conversion in the yeast Saccharomyces cerevisiae using the set of heteroallelic combinations in the ADE2 gene. Data obtained on the HIM/HIM, him/him homozygotes and HIM/him heterozygotes indicate that the him1 mutation is recessive with respect to conversion, whereas the him2, him3 and himX mutations are semidominant. Gene conversion was increased in the majority of heteroalleles of mutant diploids him1/him1. On the contrary, the him2, him3 and himX mutants have hypo-rec phenotypes on mitotic conversion. The him mutations do not affect some heteroalleles, moreover, for some heteroalleles, the effects of the him mutations was opposite. On the basis of the sum of genetical data and, particularly, of conversion event pattern in the him mutants, we suggest that him mutations analysed affect the repair pathway for mismatch correction.     

Mutants of the yeast Saccharomyces cerevisiae characterized by enhanced induced mutagenesis. III. Effect of the him mutation on the effectiveness and specificity of UF-induced mutagenesis

We have studied the influence of him1-1, him2-1, him3-1 and himX mutations on induction frequency and specificity of UV-induced adenine-dependent mutations in the yeast Saccharomyces cerevisiae. Him mutations do not render haploid cells more sensitive to the lethal action of UV-light; however, in him strains adenine-dependent mutations (ade1, ade2) were induced more frequently (1.5--2-fold), as compared to the HIM strain. An analysis of the molecular nature of ade2 mutants revealed that him1-1, him2-1 and himX mutations increase specifically the yield of transitions (AT----GC and GC----AT), whereas in the him3-1 strain the yield of transversions was enhanced as well. We suggest him mutations analysed to affect specific repair pathway for mismatch correction.     

Mutants of Saccharomyces cerevisiae supersensitive to the mutagenic effect of 6-N-hydroxylaminopurine

Yeast mutants hypersensitive to the mutagenic action of 6-N-hydroxylaminopurine (HAP) were obtained by EMS mutagenesis. One of the mutants segregated monogenically and possessed reduced capacity to utilize HAP as a purine source. A set of diploids suitable for parallel study of mutagenesis and induction of recombination, and differing in the trait of mutability after exposure to HAP ("hm" trait or HAP mutability), were constructed. It was shown that a weak recombinogenic effect of HAP is not enhanced in "hm" mutants when HAP mutability increases.     

Saccharomyces cerevisiae mutants characterized by increased induced mutagenesis. II. Genetic analysis of mutants

Induction of forward adenine-dependent (Ade+----Ade-) mutations by HAP was used to analyse genetically yeast mutants with enhanced induced mutagenesis. Three mutations studied in detail segregated as a single mendelian trait and composed independent complementation groups (HIM1, HIM2, HIM3). the him1-1 mutation was centromere-linked, the him3-1 and him2-1 mutations being not. All three mutations did not show any cross-linkage. Uracil-DNA glycosylase activity was determined in crude cell extract from wild type strain and him mutants; no detectable differences were observed.     

Aneuploidy and other genetic effects induced by hydroxyurea in Saccharomyces cerevisiae

Hydroxyurea induces mitotic gene conversion, mitotic crossing-over, reverse mutation, respiration-deficient petite mutants and aneuploidy in growing cultures of Saccharomyces cerevisiae. Evidence is presented indicating that induction rather than selection is responsible for the increase in frequency of the genetic end points measured. Complications concerning the detection of aneuploidy in the presence of other genetic effects are described, and the need for following the complete protocol for confirmation of the aneuploids in any chemical screening program is emphasized.     

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.     

The effect of sequence context on spontaneous Polzeta-dependent mutagenesis in Saccharomyces cerevisiae

The Polζ translesion synthesis (TLS) DNA polymerase is responsible for over 50% of spontaneous mutagenesis and virtually all damage-induced mutagenesis in yeast. We previously demonstrated that reversion of the lys2ΔA746 −1 frameshift allele detects a novel type of +1 frameshift that is accompanied by one or more base substitutions and depends completely on the activity of Polζ. These ‘complex’ frameshifts accumulate at two discrete hotspots (HS1 and HS2) in the absence of nucleotide excision repair, and accumulate at a third location (HS3) in the additional absence of the translesion polymerase Polη. The current study investigates the sequence requirements for accumulation of Polζ-dependent complex frameshifts at these hotspots. We observed that transposing 13 bp of identity from HS1 or HS3 to a new location within LYS2 was sufficient to recapitulate these hotspots. In addition, altering the sequence immediately upstream of HS2 had no effect on the activity of the hotspot. These data support a model in which misincorporation opposite a lesion precedes and facilitates the selected slippage event. Finally, analysis of nonsense mutation revertants indicates that Polζ can simultaneously introduce multiple base substitutions in the absence of an accompanying frameshift event.     

Mutants of Saccharomyces cerevisiae characterized by increased level of induced mutagenesis. I. Isolation and preliminary characterization of mutants

6 mutants with enhanced nitrous acid-induced reversibility of the ade2-42 allele were isolated and designated hm (high mutagenesis). Apart from sensitivity to the mutagenic exposure to nitrous acid, hm mutants were also spontaneous mutators and hypermutable under the action of UV-light and 6-N-hydroxyaminopurine. All these effects were detected not only when analysing reversibility of the ade2-42 allele, but also when scoring forward mutations in the ADE1, ADF2 genes. Gamma-mutagenesis, however, was not affected by hm mutations.     

Mapping the xrs2 and him1 genes of Saccharomyces cerevisiae by a method based on the chromosome destabilization effect

The methods for chimeric chromosomes' destabilization and the standard tetrad analysis have been used for mapping of the yeast genes XRS2 and HIM1. The genes are localized in the right arm of chromosome IV in the following order: XRS2-31cM-ADE8-28.5cM-HIM1.     

Mutagenic specificity of the base analog 6-N-hydroxylaminopurine in the LYS2 gene of yeast Saccharomyces cerevisiae

We used the LYS2 gene mutational system to study mutation specificity of the base analog 6-N-hydroxylaminopurine (HAP) in yeast. We characterized phenotypes of mutations using codon-specific nonsense suppressors and the test employing inactivation of the release factor Sup35 due to overexpression and formation of prion-like derivative [PSI]. We have shown that HAP induces predominantly nonsense mutations. While the tests using codon-specific nonsense-suppressors allowed to identify only about 50% of nonsense-mutations, all the nonsense-mutations were identified in the test with defective Sup35. We determined and analyzed the spectrum of HAP-induced nucleotide changes in two regions of the gene. HAP induces predominantly GC-->AT transitions in a hotspots of a central position of trinucleotide GGA or AGG. Directionality of these transitions is consistent with the idea that initial dHAPMP incorporation in the leading strand is more genetically dangerous than in lagging DNA strand. We revealed a specific context inhibitory for HAP mutagenesis, a "T" in -1 position to mutation site.     

The effect of 2-micron DNA on survival and mutagenesis in Saccharomyces cerevisiae

Strains of Saccharomyces cerevisiae, with and without endogenous 2-microns DNA, were studied in experiments designed to determine the effect of this plasmid on survival and mutagenesis in yeast. Comparison of the two strains exposed to ultraviolet light, 4-nitroquinoline oxide, or methyl methanesulfonate (MMS), revealed that the presence of 2-microns DNA slightly enhanced survival after exposure to each agent. Spontaneous frequencies of mutations (histidine reversion, canavanine resistance, and mitochondrial petites, but not adenine auxotrophy) were reduced by the presence of 2-microns DNA. MMS-induced His+ reversion was weak, and both strains responded similarly. No difference was found between the two strains when induced forward mutation to canavanine resistance was examined. The extent of induction of mitochondrial petites was about the same in both strains. Therefore, it appears that under these experimental conditions with these mutagens, 2-microns DNA has an effect on spontaneous mutation and survival after DNA damage but not on induced mutagenesis in S. cerevisiae.     

The effect of the erg26-1 mutation on the regulation of lipid metabolism in Saccharomyces cerevisiae

A temperature-sensitive Saccharomyces cerevisiae mutant harboring a lesion in the ERG26 gene has been isolated. ERG26 encodes 4alpha-carboxysterol-C3 dehydrogenase, one of three enzymatic activities required for the conversion of 4,4-dimethylzymosterol to zymosterol. Gas chromatography/mass spectrometry analyses of sterols in this mutant, designated erg26-1, revealed the aberrant accumulation of a 4-methyl-4-carboxy zymosterol intermediate, as well as a novel 4-carboxysterol. Neutral lipid radiolabeling studies showed that erg26-1 cells also harbored defects in the rate of biosynthesis and steady-state levels of mono-, di-, and triglycerides. Phospholipid radiolabeling studies showed defects in the rate of biosynthesis of both phosphatidic acid and phosphatidylinositol. Biochemical studies revealed that microsomes isolated from erg26-1 cells contained greatly reduced 4alpha-carboxysterol-C3 dehydrogenase activity when compared with microsomes from wild type cells. Previous studies have shown that loss of function mutations in either of the fatty acid elongase genes SUR4/ELO3 or FEN1/GNS1/ELO2 can "bypass" the essentiality of certain ERG genes (Ladeveze, V., Marcireau, C., Delourme, D., and Karst, F. (1993) Lipids 28, 907-912; Silve, S., Leplatois, P., Josse, A., Dupuy, P. H., Lanau, C., Kaghad, M., Dhers, C., Picard, C., Rahier, A., Taton, M., Le Fur, G., Caput, D., Ferrara, P., and Loison, G. (1996) Mol. Cell. Biol. 16, 2719-2727). Studies presented here have shown that this sphingolipid-dependent "bypass" mechanism did not suppress the essential requirement for zymosterol biosynthesis. However, studies aimed at understanding the underlying physiology behind the temperature-sensitive growth defect of erg26-1 cells showed that the addition of several antifungal compounds to the growth media of erg26-1 cells could suppress the temperature-sensitive growth defect. Fluorescence microscopic analysis showed that GFP-Erg26p and GFP-Erg27p fusion proteins were localized to the endoplasmic reticulum. Two-hybrid analysis indicated that Erg25p, Erg26p, and Erg27p, which are required for the biosynthesis of zymosterol, form a complex within the cell.     

Selective autophagy regulates insertional mutagenesis by the Ty1 retrotransposon in Saccharomyces cerevisiae

Macroautophagy (autophagy) is a bulk degradation system for cytoplasmic components and is ubiquitously found in eukaryotic cells. Autophagy is induced under starvation conditions and plays a cytoprotective role by degrading unwanted cytoplasmic materials. The Ty1 transposon, a member of the Ty1/copia superfamily, is the most abundant retrotransposon in the yeast Saccharomyces cerevisiae and acts to introduce mutations in the host genome via Ty1 virus-like particles (VLPs) localized in the cytoplasm. Here we show that selective autophagy downregulates Ty1 transposition by eliminating Ty1 VLPs from the cytoplasm under nutrient-limited conditions. Ty1 VLPs are targeted to autophagosomes by an interaction with Atg19. We propose that selective autophagy safeguards genome integrity against excessive insertional mutagenesis caused during nutrient starvation by transposable elements in eukaryotic cells.     

RAD18 gene product of yeast Saccharomyces cerevisiae controls mutagenesis induced by hydrogen peroxide

Within eukaryotes, tolerance to DNA damage is determined primarily by the repair pathway controlled by the members of the RAD6 epistasis group. Genetic studies on a yeast Saccharomyces cerevisiae model showed that the initial stage of postreplication repair (PRR), i.e., initiation of replication through DNA damage, is controlled by Rad6-Rad18 ubiquitin-conjugating enzyme complex. Mutants of these genes are highly sensitive to various genotoxic agents and reduce the level of induced mutagenesis. In this case, the efficiency of mutagenesis suppression depends on the type of damage. In this study we showed that DNA damage induced by hydrogen peroxide at the same mutagen doses causes significantly more mutations and lethal events in the rad18 mutant cells compared to control wild-type cells.     

Genomic instability induced by mutations in Saccharomyces cerevisiae POL1

Mutations of chromosome replication genes can be one of the early events that promote genomic instability. Among genes that are involved in chromosomal replication, DNA polymerase alpha is essential for initiation of replication and lagging-strand synthesis. Here we examined the effect of two mutations in S. cerevisiae POL1, pol1-1 and pol1-17, on a microsatellite (GT)(16) tract. The pol1-17 mutation elevated the mutation rate 13-fold by altering sequences both inside and downstream of the (GT)(16) tract, whereas the pol1-1 mutation increased the mutation rate 54-fold by predominantly altering sequences downstream of the (GT)(16) tract in a RAD52-dependent manner. In a rad52 null mutant background pol1-1 and pol1-17 also exhibited different plasmid and chromosome loss phenotypes. Deletions of mismatch repair (MMR) genes induce a differential synergistic increase in the mutation rates of pol1-1 and pol1-17. These findings suggest that perturbations of DNA replication in these two pol1 mutants are caused by different mechanisms, resulting in various types of mutations. Thus, mutations of POL1 can induce a variety of mutator phenotypes and can be a source of genomic instability in cells.     

Genetic analysis of spontaneous and 6-N-hydroxylaminopurine and propiolactone induced Adp+ mutants in Saccharomyces yeasts

652 spontaneous and 6-N-hydroxylaminopurine and propiolactone-induced mutants were obtained in yeast. 598 of them were LYS2 mutants. Detailed genetic analysis of the mutants was performed, including analysis of growth pattern on lysineless medium, suppressibility by nonsense suppressors of three types and localization on the recombination map of the LYS2 gene. Mutants induced by different agents were different for all these criteria, except for distribution among the map regions.     

The effect of the cdc9 mutation on premeiotic DNA synthesis in the yeast Saccharomyces cerevisiae

A diploid homozygous for cdc9, a conditional mutation defective in DNA ligase [2], has been used to investigate the role of this enzyme in premeiotic DNA synthesis. The cdc9 ligase has the same effect on premeiotic as on mitotic DNA synthesis and at the restrictive temperature the newly synthesized DNA is recovered in small fragments. A difference has been observed, however, between meiotic and mitotic cells, namely in their ability to join together these fragments on return to the permissive temperature. In mitotic cells this can be readly demonstrated within 50 min, whereas in contrast little joining was detected in meiotic cells, even after 2 h at the permissive temperature.     

Identification of pathways controlling DNA damage induced mutation in Saccharomyces cerevisiae

Mutation in response to most types of DNA damage is thought to be mediated by the error-prone sub-branch of post-replication repair and the associated translesion synthesis polymerases. To further understand the mutagenic response to DNA damage, we screened a collection of 4848 haploid gene deletion strains of Saccharomyces cerevisiae for decreased damage-induced mutation of the CAN1 gene. Through extensive quantitative validation of the strains identified by the screen, we identified ten genes, which included error-prone post-replication repair genes known to be involved in induced mutation, as well as two additional genes, FYV6 and RNR4. We demonstrate that FYV6 and RNR4 are epistatic with respect to induced mutation, and that they function, at least partially, independently of post-replication repair. This pathway of induced mutation appears to be mediated by an increase in dNTP levels that facilitates lesion bypass by the replicative polymerase Pol delta, and it is as important as error-prone post-replication repair in the case of UV- and MMS-induced mutation, but solely responsible for EMS-induced mutation. We show that Rnr4/Pol delta-induced mutation is efficiently inhibited by hydroxyurea, a small molecule inhibitor of ribonucleotide reductase, suggesting that if similar pathways exist in human cells, intervention in some forms of mutation may be possible.