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).     

The genetic control of spontaneous and UV-induced mitotic intrachromosomal recombination in the fission yeast Schizosaccharomyces pombe

An artificially created non-tandem heteroallelic duplication was constructed to assay mitotic intrachromosomal recombination in Schizosaccharomyces pombe. Two classes of recombinants could be distinguished: deletion-types, in which one copy of the duplicated sequence and the intervening sequence were lost, and conversion-types which retained the duplication. For spontaneous recombination, compared to wild-type cells, a rad22 mutant (corresponding to a Saccharomyces cerevisiae rad52 mutant) had wild-type levels of deletion-types, but was hypo-recombinant for conversion-types; rad16 (S. cerevisiae rad1), rad22 rad16 (S. cerevisiae rad52 rad1) and swi10 (S. cerevisiae rad10) mutants were hyper-recombinant for both types; rad22 swi10 (S. cerevisiae rad52 rad10) mutants were hypo-recombinant for both types; rhp51 (S. cerevisiae rad51) and rhp54 (S. cerevisiae rad54) mutants were hyper-recombinant for deletion-types, but almost completely lacked conversion-types. For wild-type cells, UV-irradiation induced both types of recombinant, but mainly conversion-types. All of the mutants lacked UV-induced recombination. Received: 10 June 2000 / Accepted: 22 June 2000     

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.     

A rapid method to monitor repair and mis-repair of DNA double-strand breaks by using cell extracts of the yeast Saccharomyces cerevisiae

We present a rapid in vitro method to scan the repair of DNA double-strand breaks (DSBs). A DSB was introduced at the EcoRI site within the lacZ gene of the plasmid pUC18 and the plasmid was exposed to cellular extracts from a wild-type repair-competent (RAD) and a mutant (rad52Δ) strain of the yeast Saccharomyces cerevisiae. The fidelity of rejoining was determined by the expression of the lacZ gene after bacterial transformation with the treated plasmid. A cellular extract from the yeast S. cerevisiae was found to be capable of rejoining DNA DSBs. Breaks at the EcoRI site were rejoined by extracts from both wild-type and mutant strains to form circular plasmids with almost equal efficiency. However, the fidelity of rejoining was lower for the rad52Δ extract than for normal wild-type. Received: 2 September /2 November 1997     

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.     

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.     

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.     

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.     

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     

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.     

Formation and stability of linear plasmids in a recombination deficient strain of yeast

Summary Natural termini from macronuclear DNA of the ciliated protozoans Tetrahymena thermophila and Oxytricha fallax can support telomere formation in yeast. However, plasmids carrying these ciliate termini are modified by the addition of DNA which hybridizes to the synthetic oligonucleotide poly [d(C-A)], a sequence which also hybridizes to terminal restriction fragments from yeast chromosomes but not to Tetrahymena or Oxytricha macronuclear DNAs. Thus, in yeast, the creation of new telomeres on ciliate termini involves the acquisition of yeast-specific terminal sequences presumably by either recombination or non-templated DNA synthesis. The RAD52 gene is required for the majority of yeast mitotic and meiotic recombination events. Moreover, the absence of an active RAD52 gene product results in high rates of chromosome loss. Here we demonstrate that terminal restriction fragments from Tetrahymena macronuclear ribosomal DNA (rDNA) support the formation of modified telomeres in a yeast strain carrying a defect in the RAD52 gene. Moreover, linear plasmids bearing these modified ciliate termini are stably propagated in rad52 ^– cells.     

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.     

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.     

Genetic and molecular analysis of REC114, an early meiotic recombination gene in yeast

Four new meiotic recombination genes were previously isolated by selecting for mutations that rescue the meiotic lethality of rad52 spo13 strains. One of these genes, REC114, is described here, and the data confirm that REC114 is a meiosis-specific recombination gene with no detectable function in mitosis. REC114 is located on chromosome XIII approximately 4,9 cM from CIN4. The nucleotide sequence reveals an open reading frame of 1262 bp, consensus intron splice sites close to the 3 end, and indicates that the second exon codes for only seven amino acids. In the promoter region, a URS1 consensus sequence (TGGGCGGCTA), identical to the URS1 found in the promoter of SPO16, is present 93 bp upstream of the translation start site. Northern-blot hybridization demonstrates that REC114 is transcribed only during meiosis and that it is not expressed in the absence of the IME1 gene product, even when IME2 is constitutively expressed.     

Energy-dependent mitochondrial mutagenicity of antibacterial ofloxacin and its recombinogenic activity in yeast

Ofloxacin, a specific inhibitor of bacterial topoisomerase II, is known to inhibit the growth of yeast cells and to induce rho ^– mutants in the yeast S. cerevisiae. The frequency of ofloxacin-induced petite mutants under non-growth conditions was found to be strongly diminished when the cells were depleted in intramitochondrial ATP. Under optimal conditions of mitochondrial mutagenesis the drug induced mitotic recombination and reverse mutation in diploid strains but failed to cure either killer plasmids or the 2 m DNA of dividing cells. The sensitivity to ofloxacin of the strains deficient in the DNA strandbreak repair pathway (rad52) was significantly higher then that of the wild-type strains and of the mutants deficient in excision or mutagenic DNA repair. The results are compatible with the idea that the cytotoxic and genetic activity of ofloxacin in yeast probably results from the inhibited DNA ligation function of topoisomerase II creating DNA breaks that are reparable through the recombination repair pathway.     

RAD59 and RAD1 cooperate in translocation formation by single-strand annealing in Saccharomyces cerevisiae

Studies in the budding yeast, Saccharomyces cerevisiae, have demonstrated that a substantial fraction of double-strand break repair following acute radiation exposure involves homologous recombination between repetitive genomic elements. We have previously described an assay in S. cerevisiae that allows us to model how repair of multiple breaks leads to the formation of chromosomal translocations by single-strand annealing (SSA) and found that Rad59, a paralog of the single-stranded DNA annealing protein Rad52, is critically important in this process. We have constructed several rad59 missense alleles to study its function more closely. Characterization of these mutants revealed proportional defects in both translocation formation and spontaneous direct-repeat recombination, which is also thought to occur by SSA. Combining the rad59 missense alleles with a null allele of RAD1, which encodes a subunit of a nuclease required for the removal of non-homologous tails from annealed intermediates, substantially suppressed the low frequency of translocations observed in rad1-null single mutants. These data suggest that at least one role of Rad59 in translocation formation by SSA is supporting the machinery required for cleavage of non-homologous tails.     

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.     

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.     

A newly identified DNA ligase of Saccharomyces cerevisiae involved in RAD52-independent repair of DNA double-strand breaks

Eukaryotic DNA ligases are ATP-dependent DNA strand-joining enzymes that participate in DNA replication, repair, and recombination. Whereas mammalian cells contain several different DNA ligases, encoded by at least three distinct genes, only one DNA ligase has been detected previously in either budding yeast or fission yeast. Here, we describe a newly identified nonessential Saccharomyces cerevisiae gene that encodes a DNA ligase distinct from the CDC9 gene product. This DNA ligase shares significant amino acid sequence homology with human DNA ligase IV; accordingly, we designate the yeast gene LIG4. Recombinant LIG4 protein forms a covalent enzyme-AMP complex and can join a DNA single-strand break in a DNA/RNA hybrid duplex, the preferred substrate in vitro. Disruption of the LIG4 gene causes only marginally increased cellular sensitivity to several DNA damaging agents, and does not further sensitize cdc9 or rad52 mutant cells. In contrast, lig4 mutant cells have a 1000-fold reduced capacity for correct recircularization of linearized plasmids by illegitimate end-joining after transformation. Moreover, homozygous lig4 mutant diploids sporulate less efficiently than isogenic wild-type cells, and show retarded progression through meiotic prophase I. Spore viability is normal, but lig4 mutants appear to produce a higher proportion of tetrads with only three viable spores. The mutant phenotypes are consistent with functions of LIG4 in an illegitimate DNA end-joining pathway and ensuring efficient meiosis.     

Cloning by genetic complementation and restriction mapping of the yeast HEM1 gene coding for 5-aminolevulinate synthase

Summary We have cloned the structural gene HEM1 for 5-aminolevulinate (ALA) synthase from Saccharomyces cerevisiae by transformation and complementation of a yeast hem1–5 mutant which was previously shown to lack ALA synthase activity (Urban-Grimal and Labbe Bois 1981) and had no immunodetectable ALA synthase protein when tested with yeast ALA synthase antiserum. The gene was selected from a recombinant cosmid pool which contained wild-type yeast genomic DNA fragments of an average size of 40 kb. The cloned gene was identified by the restauration.of growth on a non fermentable carbon source without addition of exogenous ALA. Sub cloning of partial Sau3A digests and functional analysis by transformation allowed us to isolate three independent plasmids, each carrying a 6 kb yeast DNA fragment inserted in either orientation into the single BamHI site of the vector pHCG3 and able to complement hem1–5 mutation. Analysis of the three plasmids by restriction endonucleases showed that HEM1 is contained within a 2.9 kb fragment. The three corresponding yeast trans formants present a 1, 2.5 and 16 fold increase in ALA synthase activity as compared to the wild-type strain. The gene product immunodetected in the transformant yeast cells has identical size as the wild-type yeast ALA synthase and its amount correlates well with the increase in ALA synthase activity.