In wild-type diploid cells of Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break (DSB) at the MAT locus can be efficiently repaired by gene conversion using the homologous chromosome sequences.Repair of the broken chromosome was nearly eliminated in rad52A diploids; 99%o lost the broken chromosome. However, in radSlA diploids, the broken chromosomes were repaired approximately 35% of the time. None of these repair events were simple gene conversions or gene conversions with an associated crossover; instead, they created diploids homozygous for the MAT locus and all markers in the 100-kb region distal to the site of the DSB. In rad51A diploids, the broken chromosome can apparently be inherited for several generations, as many of these repair events are found as sectored colonies, with one part being repaired and the other part having lost the broken chromosome. Similar events occur in about 2% of wild-type cells. We propose that a broken chromosome end can invade a homologous template in the absence of RAD51 and initiate DNA replication that may extend to the telomere, 100 or more kb away. Such breakinduced replication appears to be similar to recombinationinitiated replication in bacteria.Systems that repair chromosomal DNA double-strand breaks (DSBs) are of great importance to all cells. In the yeast Saccharomyces cerevisiae, the major pathway of DSB repair is through gap repair (1, 2), leading to a gene conversion that may be associated with a crossover of flanking markers. However, gene conversion is only one of several homologous and nonhomologous recombination pathways that are found in yeast (and mammalian cells) to repair chromosomal DSBs (3). Several of these pathways are relatively rare in yeast and can only be studied effectively when the primary homologous recombination pathway is eliminated. This can be accomplished by using deletions of genes, such as RAD52 or RAD51, that prevent chromosomal DSB repair by gene conversion (4, 5).Although the RAD52 and RAD51 gene products have been shown to interact in yeast in both genetic and biochemical assays (6, 7), they are quite different in their effects on recombination. RAD52 appears to be required for all homologous recombination events, including both gene conversion and single-strand annealing (3). In contrast, a deletion of RAD51 has no effect on single-strand annealing and has a much less profound effect on gene conversion events than does a rad52 deletion (5,(8)(9)(10). RAD51 protein appears to be homologous to the bacterial RecA protein (11-13) and has been shown to catalyze strand-exchange events in vitro that are similar but not identical to RecA-mediated events (6,14,15). However, our studies of DSB recombination suggest that the major role of RAD51 may be to promote recombination in regions of chromatin that are otherwise inaccessible rather than catalyzing these strand-exchange reactions per se (5). Thus, we found that an HO endonuclease-cleaved MAT locus on a plasmid could be repaired without RAD51 using an HO-insen...

In Saccharomyces cerevisiae, a large number of genes in the RA4D52 epistasis group has been implicated in the repair of chromosomal double-strand breaks and in both mitotic and meiotic homologous recombination. While most of these genes are essential for yeast mating-type (AM4T) gene switching, neither RAD50 nor XRS2 is required to complete this specialized mitotic gene conversion process. Using a galactose-inducible HO endonuclease gene to initiate AMT switching, we have examined the effect of null mutations of RAD50 and of XRS2 on intermediate steps of this recombination event. Both rad5O and xrs2 mutants exhibit a marked delay in the completion of switching. Both mutations reduce the extent of 5'-to-3' degradation from the end of the HO-created double-strand break. The steps of initial strand invasion and new DNA synthesis are delayed by approximately 30 min in mutant cells. However, later events are still further delayed, suggesting that XRS2 and RADSO affect more than one step in the process. In the rad5O xrs2 double mutant, the completion of MA4T switching is delayed more than in either single mutant, without reducing the overall efficiency of the process. The XRS2 gene encodes an 854-amino-acid protein with no obvious similarity to the Rad5O protein or to any other protein in the database. Overexpression of RAD50 does not complement the defects in xrs2 or vice versa.The genes RAD5O, RAD5, RAD52, RAD54, RAD55, and RADS7 have long been implicated in homology-dependent recombinational repair of radiation-induced DNA doublestrand breaks (DSBs) in Saccharomyces cerevisiae (for reviews, see references 16, 17, 24, and 43). Haploid strains with mutations in these genes respond in similar ways to X-and y-irradiation and show epistatic interactions when tested in pairwise combinations, a double mutant being no more sensitive than the more sensitive single one (24,41). This led to the establishment of the RAD52 epistasis group and to the view that products of these genes function in the same DNA repair pathway or represent components of a multiprotein DNA repair complex.However, careful examination of mutant phenotypes reveals differences between some members of the RAD52 epistasis group, notably, between RAD5O on one hand and RADS1 and RAD52 on the other (47). For example, while rad5O, rad5l, and rad52 mutants behave similarly in eliminating G2 repair of radiation-induced lesions in haploid cells, they show different radiation responses in diploids. Homozygous radSl and rad52 diploids are as sensitive to irradiation as haploid strains, demonstrating a lack of diploid-specific repair, whereas homozygous rad5O diploids show some increase in radiation resistance compared to haploid cells (54). These results can be interpreted to mean that radSl and rad52 mutants are completely deficient in the repair of radiation-induced DSBs (11,26,49) prevent radiation-induced intragenic recombination (1, 54) as well as most spontaneous events, especially gene conversion (1,21,27,31,37,58). Homothallic mating-type switching, a speci...

HO endonuclease-induced double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae can be repaired by the process of gap repair or, alternatively, by single-strand annealing if the site of the break is flanked by directly repeated homologous sequences. We have shown previously (J. Fishman-Lobell and J. E. Haber, Science 258:480-484, 1992) that during the repair of an HO-induced DSB, the excision repair gene RAD1 is needed to remove regions of nonhomology from the DSB ends. In this report, we present evidence that among nine genes involved in nucleotide excision repair, only RAD1 and RAD10 are required for removal of nonhomologous sequences from the DSB ends. rad1⌬ and rad10⌬ mutants displayed a 20-fold reduction in the ability to execute both gap repair and single-strand annealing pathways of HO-induced recombination. Mutations in RAD2, RAD3, and RAD14 reduced HO-induced recombination by about twofold. We also show that RAD7 and RAD16, which are required to remove UV photodamage from the silent HML locus, are not required for MAT switching with HML or HMR as a donor. Our results provide a molecular basis for understanding the role of yeast nucleotide excision repair genes and their human homologs in DSB-induced recombination and repair.Genes belonging to the epistasis group RAD3 are required for nucleotide excision repair (NER) of UV-damaged DNA in the yeast Saccharomyces cerevisiae (for a recent review, see reference 40). Some of these genes also function in other pathways of DNA metabolism. Genomic deletions of RAD3 (34) and SSL2 (RAD25) (17, 38) are lethal in haploid cells, indicating their essential role in maintaining cell viability. Experiments using conditional lethal mutations of RAD3 and SSL2 have demonstrated that their essential function is most likely their role in RNA polymerase II transcription (18,19,41). Substantiating the idea of an intimate linkage between NER and transcription were recent findings that protein products of at least RAD2, RAD3, RAD4, and SSL2 are components of or are physically associated with RNA polymerase II basal transcription factor b (5, 12).Experimental data also show that some NER genes have a role in mitotic recombination. Mutations in RAD1 and RAD10 reduce intrachromosomal recombination between directly repeated sequences and decrease the efficiency of homologous integration of linear DNA fragments and circular plasmids (30,45,47,48). The RAD1 function is also required for elevated recombination in cdc9 and top3 mutants (2, 47) as well as for mitotic recombination stimulated by RNA polymerase I-or II-dependent transcription (59, 67). The effect of rad1 and rad10 mutations on recombination seems to be rather specific in the sense that mitotic, but not meiotic, events are affected in the mutant strains (48). Moreover, only rad1 and rad10, not other NER mutants, showed defects in spontaneous recombination between directly repeated genes (47).A key to understanding a precise role of RAD1 in mitotic recombination came from our previous studies on the genetic control ...

In Saccharomyces cerevisiae, HO endonuclease-induced mating-type (MAT) switching is a specialized mitotic recombination event in which MAT sequences are replaced by those copied from a distant, unexpressed donor (HML or HMR). The donors have a chromatin structure inaccessible for both transcription and HO cleavage. Here we use physical monitoring of DNA to show that MAT switching is completely blocked at an early step in recombination in strains deleted for the DNA repair genes RAD51, RAD52, RAD54, RAD55 or RAD57. We find, however, that only RAD52 is required when the donor sequence is simultaneously not silenced and located on a plasmid. RAD51, RAD54, RAD55 and RAD57 are still required when the same transcribed donor is on the chromosome. We conclude that recombination in vivo occurs between DNA molecules in chromatin, whose structure significantly influences the outcome. RAD51, RAD54, RAD55 and RAD57 are all required to facilitate strand invasion into otherwise inaccessible donor sequences.

HO endonuclease-induced double-strand breaks (DSBs) within a direct duplication of Escherichia coli lacZ genes are repaired either by gene conversion or by single-strand annealing (SSA), with >80% being SSA. Previously it was demonstrated that the RAD52 gene is required for DSB-induced SSA. In the present study, the effects of other genes belonging to the RAD52 epistasis group were analyzed. We show that RAD51, RAD54, RAD55, and RAD57 genes are not required for SSA irrespective of whether recombination occurred in plasmid or chromosomal DNA. In both plasmid and chromosomal constructs with homologous sequences in direct orientation, the proportion of SSA events over gene conversion was significantly elevated in the mutant strains. However, gene conversion was not affected when the two lacZ sequences were in inverted orientation. These results suggest that there is a competition between SSA and gene conversion processes that favors SSA in the absence of RAD51, RAD54, RAD55 and RAD57. Mutations in RAD50 and XRS2 genes do not prevent the completion, but markedly retard the kinetics, of DSB repair by both mechanisms in the lacZ direct repeat plasmid, a result resembling the effects of these genes during mating-type (MAT) switching.