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.

Characterization of the role played by the RAD59 gene of Saccharomyces cerevisiae in ectopic recombination.

The RAD52 group of genes in the yeast Saccharomyces cerevisiae controls the repair of DNA damage by a recombinational mechanism. Despite the growing evidence for physical and biochemical interactions between the proteins of this repair group, mutations in individual genes show very different effects on various types of recombination. The RAD59 gene encodes a protein with similarity to Rad52p which plays a role in the repair of damage caused by ionizing radiation. In the present study we have examined the role played by the Rad59 protein in mitotic ectopic recombination and analyzed the genetic interactions with other members of the repair group. We found that Rad59p plays a role in ectopic gene conversion that depends on the presence of Rad52p but is independent of the function of the RecA homologue Rad51p and of the Rad57 protein. The RAD59 gene product also participates in the RAD1-dependent pathway of recombination between direct repeats. We propose that Rad59p may act in a salvage mechanism that operates when the Rad51 filament is not functional.

Genetic interactions between mutants of the 'error-prone' repair group of Saccharomyces cerevisiae and their effect on recombination and mutagenesis.

We have created an isogenic series of yeast strains that carry genetic systems to monitor different types of recombination and mutation [B. Liefshitz, A. Parket, R. Maya, M. Kupiec, The role of DNA repair genes in recombination between repeated sequences in yeast, Genetics 140 (1995) 1199-1211.]. In the present study we characterize the effect of mutations in genes of the 'error-prone' or postreplicative repair group on recombination and mutation. We show that rad5 and rad18 strains have elevated levels of spontaneous recombination, both of ectopic gene conversion and of recombination between direct repeats. The increase in recombination levels is similar in both mutants and in the rad5 rad18 double mutant, suggesting that the RAD5 and RAD18 gene products act together with respect to spontaneous recombination. In contrast, RAD5 and RAD18 play alternative roles in mutagenic repair: mutations in each of these genes elevate spontaneous forward mutation at the CAN1 locus, but when both genes are deleted, a low level of spontaneous mutagenesis is seen. The RAD5/RAD18 pathway of mutagenic repair is dependent on the REV3-encoded translesion polymerase. We analyze the interactions between the RAD5 and RAD18 gene products and other repair genes. The high recombination levels seen in rad5 and rad18 mutants is dependent on the RAD1, RAD51, RAD52, and RAD57 genes. The Srs2 helicase plays an important role in creating the recombinogenic substrate(s) processed by the RAD5 and RAD18 gene products.

Damage-induced ectopic recombination in the yeast Saccharomyces cerevisiae.

Mitotic recombination in the yeast Saccharomyces cerevisiae is induced when cells are irradiated with UV or X-rays, reflecting the efficient repair of damage by recombinational repair mechanisms. We have used multiply marked haploid strains that allow the simultaneous detection of several types of ectopic recombination events. We show that inter-chromosomal ectopic conversion of lys2 heteroalleles and, to a lesser extent, direct repeat recombination (DRR) between non-tandem repeats, are increased by DNA-damaging agents; in contrast, ectopic recombination of the naturally occurring Ty element is not induced. We have tested several hypotheses that could explain the preferential lack of induction of Ty recombination by DNA-damaging agents. We have found that the lack of induction cannot be explained by a cell cycle control or by an effect of the mating-type genes. We also found no role for the flanking long terminal repeats (LTRs) of the Ty in preventing the induction. Ectopic conversion, DRR, and forward mutation of artificial repeats show different kinetics of induction at various positions of the cell cycle, reflecting different mechanisms of recombination. We discuss the mechanistic and evolutionary aspects of these results.

Donation of information to the unbroken chromosome in double-strand break repair.

We have used transformation of yeast with linearized plasmids to study the transfer of information to the unbroken chromosome during double-strand break repair. Using a strain which carried the wild-type HIS3 allele, and a linearized plasmid which carried a mutant his3 allele, we have obtained His- transformants. In these, double-strand break repair has resulted in precise transfer of genetic information from the plasmid to the chromosome. Such repair events, we suggest, are gene conversions which entail the formation of heteroduplex DNA on the (unbroken) chromosome. If this suggestion is correct, our results reflect the spatial distribution of such heteroduplex DNA. Transfer of information from the plasmid to the chromosome was obtained at a maximal frequency of 1.5% of the repair events, and showed a dependence with distance. Transformation to His- was also obtained with a 2-kbp insertion and with a deletion of 200 bp. The latter results suggest that gene conversion of large heterologies can occur via repair of a heteroduplex DNA intermediate.

Donation: a new, facile method of gene replacement in yeast.

We describe here a new method for the introduction of non-selectable alleles into Saccharomyces cerevisiae, gene replacement by donation. This method only requires the availability of an autonomously replicating, selectable plasmid containing the allele to be introduced into yeast. The plasmid is digested at a restriction site (or sites) within this allele, and introduced into yeast by transformation. In the course of double-strand break repair, the entering plasmid donates genetic information to the chromosome, replacing the chromosomal allele in a gene conversion-like event. Gene replacement events are identified by a phenotypic screen of the transformants. When necessary, the transforming plasmid may be subsequently lost by segregation during permissive growth. We have studied several parameters affecting the utility of this protocol as a method of gene replacement. Together with our previous results, the results show gene replacement by donation to be a useful, facile method, yielding gene replacement in up to 1.5% of transformants.

Virus-encoded toxin of Ustilago maydis: two polypeptides are essential for activity.

Cells of Ustilago maydis containing double-stranded RNA viruses secrete a virus-encoded toxin to which other cells of the same species and related species are sensitive. Mutants affected in the expression of the KP6 toxin were characterized, and all were viral mutants. A temperature-sensitive nonkiller mutant indicated that the toxin consists of two polypeptides, 12.5K and 10K, that are essential for the toxic activity. The temperature-sensitive nonkiller mutant was affected in the expression of the 10K polypeptide, and its toxic activity was restored by the addition of the 10K polypeptide to its secreted inactive toxin. These results led to the reexamination of other mutants that were known to complement in vitro. Each was found to secrete one of the two polypeptides. Here we show for the first time that P6 toxin consists of two polypeptides that do not interact in solution, but both are essential for the toxic effect. Studies on the interaction between the two polypeptides indicated that there are no covalent or hydrogen bonds between the polypeptides. Toxin activity is not affected by the presence of 0.3 M NaCl in the toxin preparations and in the medium, suggesting that no electrostatic forces are involved in this interaction. Also, the two polypeptides do not share common antigenic determinants. The activity of the two polypeptides appears to be dependent on a sequential interaction with the target cell, and it is the 10K polypeptide that initiates the toxic effect. The similarity of the U. maydis virus-encoded toxin to that of Saccharomyces cerevisiae is discussed.

The molecular weight and packaging of dsRNAs in the mycovirus from Ustilago maydis killer strains.

The mycoviruses of Ustilago maydis killer strains are isometric, 43 nm in diameter, and contain several dsRNA segments designated heavy (H), medium (M), and light (L) according to their relative size. To determine the number of dsRNA segments per virion, major sedimenting and density components were isolated, their molecular weights determined from hydrodynamic properties, and their dsRNA contents determined by electron microscopy and/or polyacrylamide gel electrophoresis. The H dsRNA segments of 2.9, 3.1, and 4.2 x 10(6) daltons are separately encapsidated in isometric capsids that band in CsCI at 1.383, 1.394, and 1.418 g/cm8, respectively. The P1 strain contains the 3.1 and 4.2 x 10(6)-dalton segments, and the 3103 strain contains the 2.9 and 4.2 x 106-dalton segments. The T-4 strain contains the 3.1 x 106-dalton H segment and two M segments of 0.67 and 0.60 x 10(6) daltons. The H segments are separately encapsidated in virions which banded at 1.394 g/cm8, whereas the M segments are encapsidated in sets of one, two, or three in virions which banded at 1.314, 1.341, and 1.370 g/cm8. Molecular weights of 9.8 and 13.0 x 106 daltons were calculated for empty capsids (pCsCl = 1.278 g/cm8) and capsids containing the 3.1 x 10(6)-dalton dsRNA segments (pCsCl = 1.394 g/cm8). Analysis of components that banded at other densities in CsCl were consistent with the hypothesis that the banding pattern is the result of the encapsidation of a finite number of dsRNA segments in a capsid of 9.8 x 106 daltons. Although one to three M dsRNA segments were encapsidated in a single virion, no particles were detected with more than one H dsRNA segment per virion.

The killer phenomenon in Ustilago: electron microscopy of the dsRNA encapsidated in individual virus particles.

From earlier studies with the Ustilago maydis virus and other dsRNA viruses it is known that discrete dsRNA segments typical of each virus are obtained by extraction. A variation exists with respect to the encapsidation of these segments among different viruses. The encapsidation of the genome in individual particles of the Ustilago virus was examined by electron microscopy after disruption of virus particles. The study included the P6 wild-type and 2 mutants containing only part of the genome. The results indicate that most virus particles of the wild-type and the mutants contain up to 12-14 X 10(6) daltons of dsRNA. Since the largest extracted molecule is 3.2 X 10(6) D these findings suggest that an individual particle may contain more than one segment of dsRNA. Free linear molecules that exceed in size the extracted segments were also found following the disruption of each of the 3 virus types examined. Thus, the viral genome seen segmented after extraction is organized as a concatamer in the capsid and each virus particle can contain an entire viral genome consisting of each type of the segments seen after extraction, a repeat of a single segment or a random assortment. In each case the information may be organized as a concatamer.