Isolation of the RAD18 gene of Saccharomyces cerevisiae and construction of rad18 deletion mutants

Summary The RAD18 gene of Saccharomyces cerevisiae is involved in mutagenic DNA repair. We describe its isolation from a yeast library introduced into the centromeric YCp50 vector, a low copy number plasmid. The insert was sublconed into YCp50 and into the multicopy YRp7 plasmid. RAD18 is not toxic when present in multiple copies but the UV survival response indicates an heterogeneity in the cell population, a fraction of it being more sensitive. A DNA segment, close to RAD18, is toxic on the multicopy plasmid and may correspond to the tRAN sup61 known to be tightly linked to RAD18. Chromosomal deletions of RAD18 were constructed. The gene is not essential and the deleted strains have the properties of single site mutants. Thus, RAD18 appears to be essentially involved in DNA repair metabolism.     

Repair of pyrimidine dimers in radiation-sensitive mutants rad3, rad4, rad6 and rad9 of Saccharomyces cerevisiae.

The ability to remove ultraviolet (UV)-induced pyrimidine dimers was examined in four radiation-sensitive mutants of Saccharomyces cerevisiae. The susceptibility of DNA from irradiated cells to nicking by either the T4 UV-endonuclease or an endonuclease activity found in crude extracts of Micrococcus luteus was used to measure the presence of dimers in DNA. The rad3 and rad4 mutants are shown to be defective in dimer excision whereas the rad6 and rad9 mutants are proficient in dimer excision.     

Transformation and recombination in rad mutants of Saccharomyces cerevisiae

Summary Disruption/deletion mutations in genes of the RAD52 epistasis group of Saccharomyces cerevisiae were examined for their effects on recombination between single-and double-stranded circular DNA substrates and chromosomal genes in a transformation assay. In rad50 mutants there was a small reduction in recombination with single-stranded DNA at the leu2-3, 112 allele; in addition there was an almost complete elimination of recombination at trpl-1 for both single- and double-stranded DNA. Reintroduction of a wild-type RAD50 gene on a replicating plasmid carrying CEN4 restored recombinational competence at trpl-1, indicating that rad50 is defective in gene replacement of this allele. In rad52 mutants a reduction of 30%-50% in recombination involving either single- or double-stranded circular DNA was observed in each experiment when compared to the wild type. This reduction of recombination in rad52 mutants was similar for recombination at the ura352 mutant locus where only integration events have been observed, and at the trpl-1 mutant locus, where recombination occurs predominantly by gene replacement. Neither the rad54 nor the rad57 mutations had a significant effect on recombination with single- or double-stranded DNA substrates.     

Genome instability in rad54 mutants of Saccharomyces cerevisiae

The RAD54 gene of Saccharomyces cerevisiae encodes a conserved dsDNA-dependent ATPase of the Swi2/Snf2 family with a specialized function during recombinational DNA repair. Here we analyzed the consequences of the loss of Rad54 function in vegetative (mitotic) cells. Mutants in RAD54 exhibited drastically reduced rates of spontaneous intragenic recombination but were proficient for spontaneous intergenic recombinant formation. The intergenic recombinants likely arose by a RAD54-independent pathway of break-induced replication. Significantly increased rates of spontaneous chromosome loss for diploid rad54/rad54 cells were identified in several independent assays. Inter estingly, the increase in chromosome loss appeared to depend on the presence of a homolog. In addition, the rate of complex genetic events involving chromosome loss were drastically increased in diploid rad54/rad54 cells. Together, these data suggest a role for Rad54 protein in the repair of spontaneous damage, where in the absence of Rad54 protein, homologous recombination is initiated but not properly terminated, leading to misrepair and chromosome loss.     

Deletion of the SRS2 gene suppresses elevated recombination and DNA damage sensitivity in rad5 and rad18 mutants of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae genes RAD5, RAD18, and SRS2 are proposed to act in post-replicational repair of DNA damage. We have investigated the genetic interactions between mutations in these genes with respect to cell survival and ectopic gene conversion following treatment of logarithmic and early stationary cells with UV- and gamma-rays. We find that the genetic interaction between the rad5 and rad18 mutations depends on DNA damage type and position in the cell cycle at the time of treatment. Inactivation of SRS2 suppresses damage sensitivity both in rad5 and rad18 mutants, but only when treated in logarithmic phase. When irradiated in stationary phase, the srs2 mutation enhances the sensitivity of rad5 mutants, whereas it has no effect on rad18 mutants. Irrespective of the growth phase, the srs2 mutation reduces the frequency of damage-induced ectopic gene conversion in rad5 and rad18 mutants. In addition, we find that srs2 mutants exhibit reduced spontaneous and UV-induced sister chromatid recombination (SCR), whereas rad5 and rad18 mutants are proficient for SCR. We propose a model in which the Srs2 protein has pro-recombinogenic or anti-recombinogenic activity, depending on the context of the DNA damage.     

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.     

Defective thymine dimer excision in radiation-sensitive mutants rad10 and rad16 of Saccharomyces cerevisiae.

Two rad mutants of yeast, rad10 and rad16, are shown to be defective in the removal of UV-induced pyrimidine dimers since DNAs obtained from irradiated cells following a post-irradiation incubation in the dark still retain UV-endonuclease-sensitive sites. Both rad10 and rad16 mutants are in the same pathway of excision-repair as the rad1, rad2, rad3 and rad4 mutants.     

A similar protein portion for two exoglucanases secreted by Saccharomyces cerevisiae

Exoglucanase (exo-1,3-β-D-glucan glycohydrolase, EC 3.2.1.56) activity secreted by Saccharomyces cerevisiae into the culture medium was separated by ion exchange chromatography into two glycoprotein isoenzymes which contributed 10% (exoglucanase I) and 90% (exoglucanase II) towards the total activity. Analysis of the “in vitro” deglycosylated products by polyacrylamide gel electrophoresis under native or denaturing conditions indicated that the protein portions of both exoglucanases exhibited identical mobility, each one consisting of two polypeptides with M _r of 47000 and 48000. The same profile was shown by the exoglucanase secreted in the presence of tunicamycin. Antibodies raised against the protein portion of exoglucanase II did react with both native exoglucanases and their deglycosylated products with a pattern indicative of immunological identity. Digestion of the “in vitro” deglycosylated products of both exoglucanases with Staphylococcus aureus V-8 protease or trypsin generated the same proteolytic fragments in each case. Only exoglucanase II was secreted by protoplasts. These and previously reported results indicate that the protein portions of both isoenzymes may be the product of the same gene (or a family of related genes), and that exoglucanase I is a product of enzyme II, modified by a process occurring beyond the permeability barrier of the cell.     

Repair of gamma-ray induced DNA strand breaks in the radiation-sensitive mutant rad18-2 of Saccharomyces cerevisiae

The repair of gamma-ray induced DNA single and double-strand breaks was looked at in wild type and rad18-2 strains of the yeast Saccharomyces cerevisiae using sucrose gradient centrifugation. It was found that rad18-2 diploid cells could repair single and double-strand breaks induced by gamma-rays. It was also found that rad18-2 cells experienced a breakup of their DNA during post-irradiation incubation to a size smaller than seen in cells just receiving irradiation. This breakup of DNA in rad18-2 cells is not degradation due to cell death since wild type cells irradiated to similar low survival levels do not show this breakup of DNA with 8 h incubation. The breakup of DNA in rad18-2 cells is not due to replication gaps being formed by synthesis on a damaged template since treatment of rad18-2 a mating type cells with alpha factor, to prevent initiation of DNA synthesis, does not prevent breakup of the DNA.     

DNA replication in a diploid strain of Saccharomyces cerevisiae homozygous for the rad6-1 mutation

The generation time of a diploid strain homozygous for the rad6-1 mutation was 160 min, and the duration of the S phase was 80 min; in the parental heterozygote, these values were 90 and 40 min, respectively. Analysis of DNA sedimentation in an alkaline sucrose gradient revealed that heterozygote high-molecular-weight DNA appeared after 60 min, and homozygote high-molecular weight DNA only after a 100-min pulse.     

Spontaneous mutagenesis in haploid and diploid Saccharomyces cerevisiae

To obtain insights into the mechanisms of spontaneous mutations in Saccharomyces cerevisiae, we have characterized the genetic alterations that inactivate either the CAN1 gene in haploid cells or heterozygously situated in diploid cells. The mutation rate in haploid cells was 9.08 x 10(-7), 100-fold lower than that in diploid cells (1.03 x 10(-4)). In haploid cells, among 69 independent CAN1 mutations, 75% were base substitutions and 22% frameshifts. The base substitutions were both transitions (33%) and transversions (42%), with G:C-->A:T and G:C-->T:A dominating. Minus frameshifts (12%) and plus frameshifts (10%) were also observed at run and non-run bases, and at A:T and G:C pairs with almost equal efficiency. An analysis of chromosome structure in diploid yeast cells indicated that allelic crossover was the predominant event followed by gene conversion and chromosome loss. We argued that genetic alterations leading to spontaneous phenotypic changes in wild-type diploid yeast cells occurred through two steps; replication-dependent alterations of bases in either allele then recombination-dependent transfer of the mutated allele to the intact one.     

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: (1) cytosine-containing photoproducts are the substances responsible for major premutational damage to DNA; (2) a fraction of the mutations may arise in the course of excision repair of UV photoproducts.     

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.     

Suppression of the Double-Strand-Break-Repair Defect of the Saccharomyces cerevisiae rad57 Mutant

The Rad51 paralogs Rad55 and Rad57 form a heterodimer required to mediate the formation and/or stabilization of the Rad51 filament. To further characterize the function of Rad55-Rad57, we used a combination of rad57 partial suppressors to determine whether the DNA repair and recombination defects of the rad57 mutant could be completely suppressed. The combination of all suppressors, elevated temperature, srs2, rad51-I345T, and mating-type (MAT) heterozygosity resulted in almost complete suppression of the rad57 mutant defect in the recruitment of Rad51 to DNA-damaged sites, as well as survival in response to ionizing radiation and camptothecin. In a physical assay to monitor the kinetics of double-strand-break (DSB)-induced gene conversion, the rad57 mutant defect was effectively suppressed by srs2 and MAT heterozygosity, but these same suppressors failed to suppress the spontaneous recombination defect. Thus the Rad55-Rad57 heterodimer appears to have a unique function in spontaneous recombination that is not essential for DSB repair. Furthermore, we investigated the currently unknown mechanism of rad57 suppression by MAT heterozygosity and found that it is independent of DNL4.     

Aberrant double-strand break repair in rad51 mutants of Saccharomyces cerevisiae

A number of studies of Saccharomyces cerevisiae have revealed RAD51-independent recombination events. These include spontaneous and double-strand break-induced recombination between repeated sequences, and capture of a chromosome arm by break-induced replication. Although recombination between inverted repeats is considered to be a conservative intramolecular event, the lack of requirement for RAD51 suggests that repair can also occur by a nonconservative mechanism. We propose a model for RAD51-independent recombination by one-ended strand invasion coupled to DNA synthesis, followed by single-strand annealing. The Rad1/Rad10 endonuclease is required to trim intermediates formed during single-strand annealing and thus was expected to be required for RAD51-independent events by this model. Double-strand break repair between plasmid-borne inverted repeats was less efficient in rad1 rad51 double mutants than in rad1 and rad51 strains. In addition, repair events were delayed and frequently associated with plasmid loss. Furthermore, the repair products recovered from the rad1 rad51 strain were primarily in the crossover configuration, inconsistent with conservative models for mitotic double-strand break repair.     

Defective excision of pyrimidine dimers and interstrand DNA crosslinks in rad7 and rad23 mutants of Saccharomyces cerevisiae

Summary Excision of pyrimidine dimers and interstrand DNA crosslinks was examined in the deletion mutants rad7-1, rad23-1, and rad7-1 rad23-1. These mutants remove pyrimidine dimers and crosslinks much less efficiently than the RAD ⁺ strains; only 30–60% of pyrimidine dimers and 25–40% of crosslinks are removed even after prolonged incubation. The rad7 and rad23 mutations may represent defects in protein factors which increase the efficiency of the nicking enzyme complex or make chromatin more accessible to the nicking activity.