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.     

Further characterization of the yeastpso4-1 mutant: interaction withrad51 andrad52 mutants after photoinduced psoralen lesions

Thepso4-1 mutant was characterized as deficient in some types of recombination, including gene conversion, crossing over, and intrachromosomal recombination. The mode of interaction betweenpso4-1 andrad51 and betweenpso4-1 andrad52 mutants indicated that thePSO4 gene belongs to theRAD52 epistasis group for strand-break repair. Moreover, the presence of thepso4-1 mutation decreased 8-MOP-photoinduced mutagenesis of therad51 andrad52 mutants. Complementation tests using heterozygous diploid strains showed that thePso4 protein might interact with theRad52 protein during repair of 8-MOP photolesions. Thepso4-1 mutant, even though defective in inter- and intea-chromosomal recombination, conserves the ability for plasmid integration of circular and linear plasmid DNA. On the other hand, similar to therad51 mutant,pso4-1 was able to incise but did not restore high-molecular-weight DNA during the repair of cross links induced by 8-MOP plus UVA. These results, together with those of previous reports, indicate that thePSO4 gene belongs to theRAD52 DNA repair group and its product participates in the DNA rejoining step of the repair of cross-link lesions, which are crucial for induced mutagenesis and recombinogenesis.     

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).     

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.     

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.     

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.     

Interactions among mutations affecting spontaneous mutation,mitotic recombination,and DNA repair in yeast

The mutant alleles mms9-1, mms13-1, or mms21-1 of Saccharomyces cerevisiae confer pleiotropic effects, including sensitivity to the alkylating agent methyl methanesulfonate, elevations in spontaneous mutation and mitotic recombination, defects in meiosis, and cross-sensitivity to radiation. We constructed double-mutant strains containing an mms mutation and a defect in either excision repair, mutagenic repair, or recombinational repair and measured the levels of spontaneous mutation and mitotic reombination. Double mutants lacking excision repair show elevations in spontaneous mutation but with predominantly unchanged levels of mitotic recombination. RAD52 function was required for the expression of the hyper-recombination phenotype of the mms9-1, mms13-1, and mms21-1 alleles; double mutants displayed the very low recombination levels characteristic of rad52 mutants. Phenotypes of double mutants containing one of the mms alleles and either of the hyper-recombination/mutator rad6-1 or rad3-102 alleles suggest that the mutagenic lesions in mms strains may not be identical to the recombinogenic lesions.     

Evidence that an endo-exonuclease controlled by the NUC2 gene functions in the induction of ‘petite’ mutations in Saccharomyces cerevisiae

Summary Defects in the RAD52 gene of the yeast Saccharomyces cerevisiae reduce the levels of the NUC2 endo-exonuclease by approximately 90% compared to the levels in wild-type strains. To examine the potential role of this nuclease in the induction of mitochondrial petite mutations, congenic RAD52 and rad52-1 haploids were subjected to treatment with ethidium bromide, a well-known inducer of these mutations. The rad52 strain showed a much higher resistance to ethidium bromide-induced petite formation than the corresponding wild-type strain. Two approaches were taken to confirm that this finding reflected the nuclease deficiency, and not some other effect attributable to the rad52-1 mutation. First, a multicopy plasmid (YEp213-10) carrying NUC2 was transformed into a RAD52 strain. This resulted in an increased fraction of spontaneous petite mutations relative to that seen for the same strain without the plasmid and sensitized the strain carrying the plasmid to peptite induction by ethidium bromide treatment. Second, a strain having a nuc2 allele that encodes a temperaturesensitive nuclease was treated with ethidium bromide at the restrictive and permissive temperatures. Petite induction was reduced under restrictive conditions. Enzyme assays revealed that the RAD52 (YEp213-10) strain had the highest level of antibody-precipitable NUC2 endo-exonuclease whereas the nuc2 and rad52 mutants had the lowest levels. Furthermore, addition of ethidium bromide to the reaction mixture stimulated the activity of the nuclease on double-stranded DNA. Peptite induction by antifolate-mediated thymine nucleotide depletion was also inhibited by inactivation of RAD52 indicating that the effect of reduced NUC2 endo-exonuclease was not restricted to ethidium bromide treatment. Taken collectively, these results indicate that the NUC2 gene product functions in the production of mitochondrial petite mutations.     

Extragenic revertants of rad50, a yeast mutation causing defects in recombination and repair

Summary The RAD50 gene in yeast is required for recombination-repair (i.e., the double strand break repair pathway) in mitosis, and for meiotic recombination and sporulation. Both of these processes are complex and seem likely to require a relatively large number of gene products. In order to help define other genes required for recombination and repair processes in yeast, we have isolated extragenic revertants of rad50-4 which restore the ability to grow in the presence of MMS. Evidence from segregation indicates the extragenic revertants fall into at least five loci. Two of them reduce sporulation and spore viability at high temperature; another mutation confers a spontaneous hyperrec phenotype on mitotic cells. Thus, at least three revertants are candidates for mutations which affect recombination functions.     

In-vitro recombination in rad and rnc mutants of Saccharomyces cerevisiae

Summary Extracts of S. cerevisiae cells can catalyze homologous recombination between plasmids in vitro. Extracts prepared from rad50, rad52 or rad54 disruption mutants all have reduced recombinational activity compared to wild-type. The rad52 and rad54 extracts are more impaired in the recombination of plasmids containing double-strand breaks than of intact plasmids, whereas rad50 extracts are deficient equally for both types of substrate. The nuclease RhoNuc (previously designated yNucR), encoded by the RNC1 (previously designated NUC2) gene and regulated by the RAD52 gene, is not required for recombination when one substrate is single-stranded but is essential for the majority of recombination events when both substrates are double-stranded. Furthermore, elimination of this nuclease restores recombination in rad52 extracts to levels comparable to those in wild-type extracts.     

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     

Spontaneous mitotic recombination inSchizosaccharomyces pomhe

Summary The UV sensitive mutantrad2-44 ofSchizosaccharomyces pomhe increases mitotic gene conversion and crossover rates about 10-fold but has little or no effect on meiotic recombination. As inrad2⁺, recombination events on different chromosomes are coincidental inrad2-44, indicating that mitotic recombination takes place in a subpopulation of competent cells. However, the coefficient of coincidence is smaller in the mutant, whereas recombination rates among the competent cells are the same as inrad2⁺. This suggests thatrad2-44 increases mitotic recombination by enhancing the fraction of competent cells. The rate limiting factor in spontaneous mitotic recombination inS. pomhe appears to be the size of the subpopulation of recombinationally competent cells.     

Mating-type suppression of the DNA-repair defect of the yeast rad6Δ mutation requires the activity of genes in the RAD52 epistasis group

The RAD6 gene of Saccharomyces cerevisiae is required for post-replication repair of UV-damaged DNA, UV mutagenesis, and sporulation. Here, we show that the radiation sensitivity of a MAT a rad6 strain can be suppressed by the MAT2 gene carried on a multicopy plasmid. The a1-2 suppression is specific to the RAD6 pathway, as mutations in genes required for nucleotide excision repair or for recombinational repair do not show such mating-type suppression. The a1-2 suppression of the rad6 mutation requires the activity of the RAD52 group of genes, suggesting that suppression occurs by channelling of post-replication gaps present in the rad6 mutant into the RAD52 recombinational repair pathway. The a1-2 repressor could mediate this suppression via an enhancement in the expression, or the activity, of recombination genes.     

The influence of mutation rad57-1 on the fidelity of DNA double-strand gap repair in Saccharomyces cerevisiae

The role of the RAD57 gene in double-strand gap (DSG) repair has been examined. The repair of a linearized plasmid, bearing a DSG, has been analyzed in a rad57-1 mutant of Saccharomyces cerevisiae. For effective rejoining of the ends of plasmid DNA in the rad57 mutant the sequence of chromosomal DNA homologous to the DSG region is required. However, DSG repair (restoration of plasmid circularity) in rad57 cells is not accompanied by the recovery of DSGs. The DSG repair, which depends on an homologous chromosomal DNA sequence, requires the cohesive ends of DSGs. The non-cohesive-ended DSGs are repaired in rad57 cells by a pathway independent of the homologous recombination between chromosomal and plasmid DNA. We presume that the rad57-1 mutation is connected with the inhibition of DNA repair synthesis, required for filling the DSG. This situation produces a condition of “homology-dependent ligation”, the alternative minor mechanism of recombinational DSG repair, that takes place in mutant cells. A molecular model for “homology-dependent ligation” in rad57 cells is proposed. Received: 26 March / 29 October 1998     

Genetic effects of photoactivated psoralens during meiosis in DNA repair mutantpso3-1 ofSaccharomyces cerevisiae

The influence of the DNA repair genePSO3 on photoactivated psoralen-induced meiotic recombination, gene conversion, reverse mutation, and on survival, was assayed in diploid strains ofSaccharomyces cerevisiae homozygous for the wild-type or thepso3-1 mutant allele. Sporulation was normal in thepso3-1 diploid. Wild-type and mutant strains had the same sensitivity to photoactivated monofunctional psoralen (3-CPs+UVA) in meiosis-uncommitted and meiosis-committed stages. The mutant showed higher sensitivity to photoactivated bifunctional psoralen (8-MOP+UVA) during all stages of the meiotic cycle. Mutation induction by 3-CPs+UVA or 8-MOP+UVA in meiosis-committed cells revealed no significant differences between wild-type and thepso3-1 mutant. The status of thePSO3 gene has no influence on the kinetics of induction of gene conversion and crossing-over after 3-CPs+UVA treatment in meiosis-committed cells: gene conversion was blocked while recombination was induced. After treatment with 8-MOP+UVA gene conversion was also blocked in both strains while crossing-over could only be observed in meiosis-committed wild-type cells.     

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     

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.     

Homologous recombination in the fission yeast Schizosaccharomyces pombe: different requirements for the rhp51+, rhp54+ and rad22+ genes

The Schizosaccharomyces pombe rhp51 ⁺ , rad22 ⁺ and rhp54 ⁺ genes are homologous to RAD51, RAD52 and RAD54 respectively, which are indispensable in the recombinational repair of double-strand breaks (DSBs) in Saccharomyces cerevisiae. The rhp51Δ and rhp54Δ strains are extremely sensitive to ionizing radiation; the rad22Δ mutant turned out to be much less sensitive. Homologous recombination in these mutants was studied by targeted integration at the leu1-32 locus. These experiments revealed that rhp51Δ and rhp54Δ are equally impaired in the integration of plasmid molecules (15-fold reduction), while integration in the rad22Δ mutant is only reduced by a factor of two. Blot-analysis demonstrated that the majority of the leu⁺ transformants of the wild-type and rad22Δ strains have integrated one or more copies of the vector. Gene conversion events were observed in less than 10% of the transformants. Interestingly, the relative contribution of gene conversion events is much higher in a rhp51Δ and a rhp54Δ background. Meiotic recombination is hardly affected in the rad22Δ mutant. The rhp51Δ and rhp54Δ strains also show minor deficiencies in this type of recombination. The viability of spores is 46% in the rad22Δ strain and 27% in the rhp54Δ strain, as compared with wild-type cells. However, in the rhp51Δ mutant the spore viability is only 1.7%, suggesting an essential role for Rhp51 in meiosis. The function of Rhp51 and Rhp54 in damage repair and recombination resembles the role of Rad51 and Rad54 in S. cerevisiae. Compared with Rad52 from S. cerevisiae, Rad22 has a much less prominent role in the recombinational repair pathway in S. pombe. Received: 20 July 1996     

Inhibition of thymidylate biosynthesis induces mitotic unequal sister chromatid recombination in Saccharomyces cerevisiae

Summary Inhibition of thymidylate biosynthesis has been found to induce deletion of a LEU2 insert from the ribosomal DNA gene cluster of haploid strains of Saccharomyces cerevisiae. Loss of the insert was detected phenotypically by the enhanced production of both sectored (leu⁺/leu^–) and non-sectored (leu^–) colonies. Hybridization patterns obtained by Southern blot analysis of DNA from the leu⁺ and leu^– sectors were consistent with the occurrence of unequal sister chromatid recombination. The induction of sectored colonies was prevented by the rad52-1 mutation but not by defects in RAD6. However, the formation of non-sectored leu^– colonies was induced by thymidylate depletion in both rad52-1 and rad6 strains.