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...