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.     

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).     

Interactions of the RAD7 and RAD23 excision repair genes of Saccharomyces cerevisiae with DNA repair genes in different epistasis groups

Summary The RAD7 and RAD23 genes of S. cerevisiae affect the efficiency of excision repair of UV-damaged DNA. We have examined the UV survival of strains carrying the rad7 or rad23 deletion mutation in combination with deletion mutations in genes affecting different DNA repair pathways. As expected, the rad7 and rad23 mutations interact epistatically with the excision repair defective rad1 mutation, and synergistically with the rad6 and rad52 mutations that affect the postreplication repair and recombinational repair pathways, respectively. However, the rad7rad6 and the rad23rad6 mutants exhibit the same level of UV sensitivity as the radlrad6 mutant. This observation is of interest since, in contrast to the rad7 or the rad23 mutations, the rad1 mutant is very UV sensitive and highly excision defective. This observation suggests that RAD6 and RAD7 and RAD23 genes compete for the same substrate during DNA repair.     

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.     

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.     

Suppression of a mitochondrial point mutation in a tRNA gene can cast light on the mechanisms of 3′ end-processing

We used a genetic approach to study the nuclear factors involved in the biogenesis of mitochondrial tRNAs. A point mutation in the mitochondrial tRNA^Asp gene of Saccharomyces cerevisiae had previously been shown to result in a temperature-sensitive respiratory-deficient phenotype as a result of the absence of 3 end-processing of the tRNA^Asp. Analysis of mitochondrial revertants has shown that all revertants sequenced have a G-A compensatory change at position 53, which restores the hydrogen-bond with the mutated nucleotide. We then searched for nuclear suppressors to identify the nuclear gene(s) involved in mitochondrial tRNA 3 end-processing. One such suppressor mutation was further characterized: it restores tRNA^Asp maturation and growth at 36°C on glycerol medium in heterozygous diploids, but leads to a defective growth phenotype in haploids.     

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 of Saccharomyces cerevisiae is not essential for vegetative growth

Summary The gene RAD50 was located by the ability of subclones to restore the Rad⁺ phenotype following transformation of a rad50-1 mutant. Disruption of the gene was achieved by directed integration of a plasmid carrying a fragment internal to RAD50. Haploids with the disrupted gene are viable and do not differ in growth rate or plating efficiency from isogenic rad50-1 or Rad⁺ strains.     

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.     

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.     

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.     

Induced cellular resistance to ultraviolet light in Saccharomyces cerevisiae is not accompanied by increased repair of plasmid DNA

Summary Many reports show that resistance of Saccharomyces cerevisiae to a large UV dose can be enhanced by pre-induction with a smaller one given some hours before. This work tests if such increased cell survival is associated with increased DNA repair on UV damaged plasmid transformed into yeast. There was no change in transformation efficiency of UV-damaged plasmid DNA under conditions where RAD cell survival increased 5-fold, and where rad1-1 and rad6-1 survival increased 2-fold. It is concluded that DNA repair activity involving the RAD6 and RAD3 pathways is either not inducible or is unable to work on plasmid DNA. It is suggested that the enhancement of cellular survival detected may be based on changes in cell-cycle behaviour which permit cells generally proficient in repair a greater chance to recover.     

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.     

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.     

Genetic and physical interactions between Schizosaccharomyces pombe Mcl1 and Rad2, Dna2 and DNA polymerase α: evidence for a multifunctional role of Mcl1 in DNA replication and repair

Schizosaccharomyces pombe rad2 is involved in Okazaki fragments processing during lagging-strand DNA replication. Previous studies identified several slr mutants that are co-lethal with rad2 and sensitive to methyl methanesulfonate as single mutants. One of these mutants, slr3-1, is characterized here. Complementation and sequence analyses show that slr3-1 (mcl1-101) is allelic to mcl1⁺, which is required for chromosome replication, cohesion and segregation. mcl1-101 is temperature-sensitive for growth and is highly sensitive to DNA damage. mcl1 cells arrest with 2C DNA content and chromosomal DNA double-strand breaks accumulate at the restrictive temperature. Mcl1p, which belongs to the Ctf4p/SepBp family, interacts both genetically and physically with DNA polymerase . Mutations in rhp51 and dna2 enhance the growth defect of the mcl1-101 mutant. These results strongly suggest that Mcl1p is a functional homologue of Saccharomyces cerevisiae Ctf4p and plays a role in lagging-strand synthesis and Okazaki fragment processing, in addition to DNA repair.     

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.     

Biased inheritance of optional insertions following mitochondrial genome recombination in the basidiomycete fungus Coprinus cinereuss

Summary Two mitochondrial genomes of Coprinus cinereus, H and J, were found to have alternative 1.23 kb insertions. Using the Neurospora crassa cytochrome oxidase-1 (co-1) gene as a probe, the J insertion site was shown to be located within the Coprinus co-1 gene, whereas the H insertion was some 2 kb distant. The insertions showed biased inheritance following mitochondrial genome recombination. Recombination between H and J genomes was detected using the mitochondrial gene mutations acu-10, which causes a cytochrome oxidase defect, and cap-1, which confers chloramphenicol resistance. Fourteen of fifteen independently derived recombinants for these two genes were shown to have both DNA insertions. In a second series of H x J crosses, intragenic recombination between different cap-1 alleles was detected. These mutations are assumed to be in the large ribosomal RNA gene some 6 kb distant from the nearest insertion site. Each of eight independently derived cap-1 ⁺ recombinants had both DNA insertions. Despite their similar size and similar behaviour following recombination the insertions do not share extensive sequence homology.     

Genetic and cytological characterization of the RecA-homologous proteins Rad51 and Dmc1 of Schizosaccharomyces pombe

The Schizosaccharomyces pombe rad51⁺ and dmc1⁺ genes code for homologues of the Escherichia coli recombination protein RecA. Deletion of rad51⁺ causes slow growth, retardation of cell division and a decrease in viability. rad51 cells have a defect in mating-type switching. The DNA modification at the mating-type locus required for mating-type switching contributes to slow growth in the rad51 mutant. Cell mating is reduced in crosses homozygous for rad51. Ectopic expression of the dmc1⁺ gene allowed us to demonstrate that the reduction in meiotic recombination in dmc1 mutants is not caused by a disturbance of rad24 expression from the dmc1-rad24 bicistronic RNA. We describe the functional defects of terminally epitope-tagged Dmc1 and Rad51 and discuss it in terms of protein interaction. Presumptive Rad51 and Dmc1 foci were detected on spreads of meiotic chromatin.     

The use of a double-marker shuttle vector to study DNA double-strand break repair in wild-type and radiation-sensitive mutants of the yeast Saccharomyces cerevisiae

An episomal DNA vector (YpJA18), encoding two selectable recombinant yeast genes (TRP1, URA3), was constructed to assess the fidelity of DNA repair in haploid repair-competent (RAD) wild-type yeast and several radiation-sensitive mutants. Either a DNA double-strand break (DSB) or a double-strand gap of 169 bp (DSG) was introduced by restriction enzymes in-vitro within the coding sequence of the URA3 gene of this vector. To eliminate transfer artefacts, selection was first applied for the undamaged TRP1 gene followed by counter selection for URA3 gene activity, which indicated correct repair of the DSB and DSG. Correct repair of the damaged URA3 gene was found to be about 90% in RAD cells (normalized for the expression of undamaged URA3 in TRP ⁺ transformants). Plasmids isolated from the transformants (URA ⁺ TRP ⁺) carry both unique sites (ApaI and NcoI) within the URA3 gene indicating the precise restitution of the 169-bp gap. An excision-repair-defective rad4-4 mutant repaired these lesions as correctly as RAD cells, whereas the mutants rad50-1, rad51-1 and rad54-1, proven to be defective in DSB repair and mitotic recombination, showed less than 5% correct repair of such lesions. In contrast, a representative of the RAD6 epistasis group of genes, the rev2-1 mutant which is sensitive towards UV and ionizing radiation, had a significantly reduced ability (about 20%) for the correct repair of both DSBs and DSGs.     

Some of the swi genes of Schizosaccharomyces pombe also have a function in the repair of radiation damage

Summary In Schizosaccharomyces pombe the frequency of mating-type (MT) switching is reduced by mutations in the swi genes. The ten hitherto known swi genes can be subdivided into three classes: Ia, Ib and II. Strains having swi5 (class Ib), swi9 (class II) and swi10 (class II) mutations do not only show reduced MT switching, but also exhibit an increased sensitivity to UV- and -rays. For that reason, 19 previously described rad genes were tested for their effect on MT switching. We found that swi9, rad10, rad16 and rad20 are allelic with each other indicating that the former allocation of these rad mutations to three different genes must have been erroneous. Among the remaining 16 rad genes examined, rad22 seems to be a new class II swi gene. The double mutants swi5 swi9 and swi5 swi10, but not swi9 swi10, are much more sensitive to radiation than the respective single mutants. Thus a cumulative increase in sensitivity occurs only if the mutants belong to different classes; previously the same correlation was found with regard to cumulative effects in MT switching.