A reexamination of the role of the RAD52 gene in spontaneous mitotic recombination

Summary The RAD52 gene is required for much of the recombination that occurs in Saccharomyces cerevisiae. One of the two commonly utilized mutant alleles, rad52-2, increases rather than reduces mitotic recombination, yet in other respects appears to be a typical rad52 mutant allele. This raises the question as to whether RAD52 is really necessary for mitotic recombination. Analysis of a deletion/insertion allele created in vitro indicates that the null mutant phenotype is indeed a deficiency in mitotic recombination, especially in gene conversion. The data also indicate that RAD52 is required for crossing-over between at least some chromosomes. Finally, examination of the behavior of a replicating plasmid in rad52-1 strains indicates that the frequency of plasmid integration is substantially reduced from that in wild type, a conclusion consistent with a role for RAD52 in reciprocal crossing-over. Analysis of recombinants arising in rad52-2 strains suggests that this allele may result in the increased activity of a RAD52-independent recombinational pathway.     

Cold-sensitive rad52 alleles of yeast

We have characterized two rad52 mutations that are cold-sensitive for growth on MMS agar. The mutations change residues 61 and 69, respectively, in the 504 amino-acid open reading frame. Neither mutation has a profound effect on mitotic crossing-over or on gene conversion. One has a severe deficiency in sporulation at all temperatures, while the other has a partial deficiency and reduced spore viability. Both mutants are retarded in growth on MMS agar by a high-copy plasmid expressing RAD51. Received: 18 April / 14 May 1997     

RAD58 (XRS4)—A new gene in the RAD52 epistasis group

The RAD58 (XRS4) gene of Saccharomyces cerevisiae has been previously identified as a DNA repair gene. In this communication, we show that RAD58 also encodes an essential meiotic function. The spore inviability of rad58 strains is not rescued by a spo13 mutation. The rad50 mutation suppresses spore inviability of a spo13 rad58 strain suggesting that RAD58 acts after RAD50 in meiotic recombination. The rad58-4 mutation does not prevent mitotic recombination events. Haploid rad58 cells fail to carry out G₂-repair of gamma-induced lesions, whereas rad58/rad58 diploids are able to perform some diploid-specific repair of these lesions.     

Genetic evidence for different RAD52-dependent intrachromosomal recombination pathways in Saccharomyces cerevisiae

Mutations in the RecA-like genes RAD51 and RAD57 reduce the frequency of gene conversion/reciprocal exchange between inverted repeats 7-fold. However, they enhance the frequency of deletions between direct repeats 5–12-fold. These induced deletions are RAD1- and RAD52-dependent. On the basis of these results it is proposed that there are several RAD52-dependent pathways of recombination: the recombinational repair pathway of gene conversion/reciprocal exchange dependent on RAD51 and RAD57; a RAD1-and RAD52-dependent pathway exclusively responsible for deletions that are induced in rad51 and rad57 mutants; and finally, it is possible that the gene conversion/reciprocal exchange events observed in rad51 and rad57 strains represent another RAD52-dependent recombination pathway of gene conversion/reciprocal exchange that does not require Rad51 and Rad57 functions. It is also shown that the RAD10 excision-repair gene is involved in long gene conversion tracts in homologous recombination between inverted repeats, as previously observed for RAD1. Finally, an analysis of meiotic recombination reveals that deletions are induced in meiosis 100-fold above mitotic levels, similar to intrachromosomal gene conversion/reciprocal exchange, and that, in contrast to intrachromosomal meiotic gene conversion (50% association), intrachromosomal meiotic gene conversion is not preferentially associated with reciprocal exchange (12–30% of association).     

Unequal sister-strand recombination within yeast ribosomal DNA does not require the RAD 52 gene product

Summary We have found that the RAD52 gene product, which is required for gene conversion and recombination in the yeast Saccharomyces cerevisiae, is not required for unequal mitotic sister-strand recombination.     

Effects of the rad52 gene on sister chromatid recombination in Saccharomyces cerevisiae

Summary Spontaneous and UV induced unequal mitotic sister chromatid recombination was examined in RAD+ and rad52-1 strains carrying the LEU2 gene inserted in the rDNA locus. The rad52-1 mutation does not affect spontaneous sister chromatid recombination but greatly reduces the frequency of UV induced sister chromatid recombination.     

The Saccharomyces cerevisiae RAD2 gene complements a Schizosaccharomyces pombe repair mutation

Summary Two Saccharomyces cerevisiae genes necessary for excision repair of UV damage in DNA, RAD1 and RAD2, were introduced individually, on a yeast shuttle vector, into seven Schizosaccharomyces pombe mutants — rads1, 2, 5, 13, 15,16 and 17. The presence of the cloned RAD1 gene did not affect survival of any of the S. pombe mutants. The RAD2 gene increased survival of S. pombe rad13 to near the wild-type level after UV irradiation and had no effect on any of the other mutants tested. S. pombe rad13 mutants are somewhat defective in removal of pyrimidine dimers so complementation by the S. cerevisiae RAD2 gene suggests that the genes may code for equivalent proteins in the two yeasts.     

Allele-specific suppression of a cell cycle mutant of S. cerevisiae

Summary A cell cycle (cdc) mutant of Saccharomyces cerevisiae is described which fails to complement cdc27-1 described by Hartwell et al. 1973), and is designated cdc27–47. Whereas cdc27-1 behaves as a single Mendelian gene (Hartwell et al. 1973), cdc27–47 requires the presence of an additional unlinked gene for expression of temperature-sensitivity. This gene, designated sts47, is present in some, but not all, laboratory wild-type strains. Expression of cdc27-1 is not influenced by STS47. A model for suppression is proposed involving the modification of conformation within a structural or enzyme complex.     

The RAD50 gene,a member of the double strand break repair epistasis group,is not required for spontaneous mitotic recombination in yeast

Summary Mutations in the RAD50 gene of Saccharomyces cerevisiae have been shown to reduce double strand break repair, meiotic recombination, and radiation-inducible mitotic recombination. Several different point mutations (including ochre and amber alleles) have been previously examined for effects on spontaneous mitotic recombination and did not reduce the frequency of recombination. Instead, the rad50 mutations conferred a moderate hyper-rec phenotype. This paper examines a deletion/interruption allele of RAD50 that removes 998 of 1312 amino acids and adds 1.1 kb of foreign DNA. The results clearly indicate that spontaneous mitotic recombination can occur in the absence of RAD50; in fact, the frequency of recombination is elevated over the wild-type cell. One possible interpretation of these observations is that the initiating lesion in spontaneous recombination events in mitosis might not be a double strand break.     

The mcm2-1 mutation of yeast causes DNA damage with a RAD9 requirement for repair

The minichromosome maintenance mutation, mcm2-1, has been found to synthesize damaged DNA at 35°C. Growth at this temperature rendered the mutant strain more sensitive to killing by ultraviolet irradiation. DNA damage could also be detected by pulsed-field gel electrophoresis, where a higher fraction of the DNA loaded was retained in the inserts at the wells. During the exponential phase of growth at this temperature about 50% of the cells had large buds, with the nucleus at or near the neck of the bud in most cases. The incorporation of the rad9 deletion in the mcm2-1-carrying strain caused a reduction in the percentage of large-budded cells and a moderate loss of cell viability. The results are consistent with mcm2-1 causing DNA damage leading to the arrest of cells in the S/G₂ phase of the cell cycle, which was partially dependent on the RAD9 gene product.     

DNA repair genes of Saccharomyces cerevisiae: complementing rad4 and rev2 mutations by plasmids which cannot be propagated in Escherichia coli

Summary The RAD4 gene of yeast required for the incision step of DNA excision repair and the REV2 (= RAD5) gene involved in mutagenic DNA repair could not be isolated from genomic libraries propagated in E. coli regardless of copy number of the shuttle vector in yeast. Transformants with plasmids conferring UV resistance to a rad4-4 or a rev2-1 mutant were only recovered if yeast was transformed directly without previous amplification of the gene bank in E. coli. DNA preparations from these yeast clones yielded no transformants in E. coli but retransformation of yeast was possible. This lead to the isolation of a defective derivative of the rad4 complementing plasmid. The modified plasmid was now capable of transforming E. coli but still interfered significantly with its growth.Dedicated to Prof. Dr. Fritz Kaudewitz on the occasion of his 65th birthday     

Mapping of the RIB5 gene in Saccharomyces cerevisiae using UV light as an enhancer of rad52-mediated chromosome loss

Summary Ribs mutants of S. cerevisiae are blocked at the end of the riboflavin biosynthetic pathway. Using UV light to increase rad52-mediated chromosome loss, we have assigned the rib5 mutation to chromosome II. Tetrad analysis of crosses between rib5 and other markers on chromosome II shows that the RIB5 gene is located on the right arm of this chromosome, closely linked to HIS7.     

Cloning and integrative deletion of the RAD6 gene of Saccharomyces cerevisiae

Summary Three overlapping plasmids were isolated from a YEp24 library, which restore Rad+ functions to rad6-1 and rad6-3 mutants. Different subclones were made and shown to integrate by homologous recombination at the RAD6 site on chromosome VII, thus verifying the cloned DNA segments to be the RAD6 gene and not a suppressor. The gene resides in a 1.15 kb fragment, which restores Rad⁺ levels of resistance to U.V., MMS and -rays to both rad6-1 and rad6-3 strains. It also restores sporulation ability to rad6-1 diploids.Integrative deletion of the RAD6 gene was shown not to be completely lethal to the yeast. Our results suggest that the RAD6 gene has some cell cycle-specific function(s), probably during late S phase.     

The DNA repair gene PSO3 of Saccharomyces cerevisiae belongs to the RAD3 epistasis group

Summary The mutant allele pso3-1 of Saccharomyces cerevisiae confers sensitivity to treatment with UV_365nm (UVA) light-activated mono- and bi-functional psoralens. When pso3-1 is combined in double mutants with selected rad and pso mutant alleles and subjected to 8-MOP+UVA treatment, epistatic interaction with regard to survival is observed with pso1, pso2, and rad3. With the same treatment the combination of pso3-1 with rad6 and rad52 leads to synergistic interaction. For the monofunctional agent 3-carbethoxypsoralen (3-CPs) the analysis of double mutants yields the same results as with the bifunctional 8-methoxypsoralen (8-MOP) with the exception of the pso1-1pso3-1 double mutant. Here we find an additive interaction, i.e., the sensitivities of both parental strains are summed in the double mutant, which indicates a different substrate specificity of the repair activity encoded by the PSO1 and PSO3 genes.     

Alkylation mutagenesis in Saccharomyces cerevisiae: lack of evidence for an adaptive response

Summary We have found no evidence for an adaptive response for either lethality or mutagenesis following treatment of Saccharomyces cerevisiae with N-methyl-N-nitro-N-nitrosoguanidine (MNNG). The rad6 and rad52 mutants of S. cerevisiae are highly defective in MNNG and ethyl methanesulfonate induced mutagenesis of both stationary and exponential phase cells. These and other observations indicate that the mechanisms of repair of alkylation damage and mutagenesis differ markedly between S. cerevisiae and Escherichia coli.     

Interaction of excision repair gene products and mitotic recombination functions in yeast

We have tested the ability of mutants of three additional genes in the excision repair pathway of Saccharomyces cerevisiae to suppress the hyper-recombination and rad52 double-mutant lethality phenotypes of the rad3-102 (formerly rem1-2) mutation. Such suppression has previously been been observed with mutant alleles of RAD1 and RAD4. We had hypothesized that the rad3-102 mutation created elevated levels of DNA lesions which could be processed by the products of the RAD1 and RAD4 genes into recombinogenic double-strand breaks requiring the RAD52 product for repair. In this report, we show that the RAD2, RAD7, and RAD10 genes are also necessary for this processing. We discuss our observations of varying levels of mitotic crossingover in Rem^- rad double-mutant strains.     

FLP recombinase induction of the breakage-fusion-bridge cycle and gene conversion in Saccharomyces cerevisiae

Summary A YEp chimaeric plasmid containing URA3 and SMR1 [sulfometuron methyl resistant (SM^R) allele of ILV2] as selectable markers, and the 2 m site-specific recombination FLP recognition target (FRT), was integrated at the ilv2-1 site in chromosome XIII in a cir°] haploid. Southern analysis defined two integrant structures. Structure I had URA3 distal and SMR1 proximal to FRT whereas in structure II both markers were distal to FRT. Selectable markers were stably inherited in [cir°] haploids and [cir°] diploids heterozygous for the integrant and ILV2. Approximately 14% of heterozygous [cir ⁺] diploid cells exhibited homozygotization for the distal (500 kb) ade4 marker in trans. In [cir ⁺] diploids FLP-FRT recombination resulted in the simultaneous loss of both structure II markers, whereas the structure I distal URA3 marker loss always preceded the variable loss of the proximal SMR1 marker. URA^– cells continued to segregate for loss of SMR1 until stable URA^– SM^R or URA^–SM^S cells were produced. Gene conversion was identified in stable URA^–SM^R cells that were homozygous SMR1/SMR1 but contained wild type ILV2 restriction endonuclease sites. These observations support a model based on concerted FLP-FRT action resulting from the secondary integration of native 2 m DNA followed by unequal sister chromatid exchange (USCE) within inverted FRTs. The resultant chromatid bridge resulted in a double-stand break. Fusion of the broken ends of sister chromatids generated a breakage-fusion-bridge cycle (BFBC). Repeated rounds of the BFBC resulted in proximal marker loss and the generation of additional double-strand breaks. Recombinogenic properties of the double-strand break initiated events leading to homozygotization and gene conversion.     

The DEL1 mutator gene in Saccharomyces cerevisiae does not act in trans

Summary DEL1 strains of the yeast Saccharomyces cerevisiae exhibit a high rate of deletions of the three linked genes, CYC1, OSM1, and RAD7. Classical genetic methods showed that DELI segregated as a single Mendelian gene closely linked to CYC1. In addition, genetic evidence suggested that DEL1 was both cis- and trans-dominant (Liebman et al. 1979). Molecular analysis of deletions isolated from a haploid DEL1 strain established that deletion formation was mediated by recombination between yeast transposable elements, Ty's (Liebman et al. 1981). We now report the molecular characterization of deletions isolated from diploids in the trans configuration. This analysis reveals that these deletions probably arose in a two-step process involving mitotic recombination followed by Ty-mediated deletion formation in cis.