Mating type switching in Saccharomyces cerevisiae initiates when Ho endonuclease makes a site-specific double-stranded break at MAT, the yeast mating type locus. To identify other proteins involved in this process, we examined whether extracts prepared from ho ؊ mutants contain additional factors that bind near the recognition sequence for Ho. Using an electrophoretic mobility shift assay, we isolated a chromatographic fraction that contains an activity, named YZbp, which binds to two sequences flanking the recognition sequence at MAT␣ and to one sequence overlapping it at MATa. MAT plasmids carrying mutations in the YZbp recognition sequence are cleaved by purified Ho at wild-type efficiencies in an in vitro assay. These same plasmids, however, are not cleaved by Ho inside cells, demonstrating that YZbp acts as a positive activator of in vivo cleavage. YZbp is present in all cell types, even those not undergoing mating type switching, suggesting that it has additional cellular functions.Genetic recombination is the intracellular process that moves DNA sequence information from one genomic location to another. In the yeast Saccharomyces cerevisiae, several lines of evidence indicate that recombination initiates with a doublestranded break (DSB) in DNA. First, DSBs are repaired by using gene products required for recombination (reviewed in reference 22). Second, certain meiotic segregation patterns of yeast genes are consistent with molecular models of recombination that posit a DSB initiation event (28). Third, DSBs occur at regions of the genome defined genetically as initiators of recombination (2,4,6,8,21,26,27,33,34). Fourth, DSBs introduced into DNA during meiosis stimulate nearby meiotic recombination (13). Fifth, meiotic DSBs precede and are independent of pairing between homologs, implying that DSBs initiate chromosomal pairing (3, 7). Finally, mating type switching, a specialized form of mitotic gene conversion, has been shown unambiguously to initiate with a DSB (14,15,25). While these results clearly show that DSBs play a major role in the initiation of recombination, it is important to state that other types of intermediates, such as single-stranded nicks in DNA, might perform similar functions.Given the important role of DSBs in the initiation of recombination, it seems likely that their formation is a highly regulated process that depends on multiple gene products. The goal of this study was to identify factors involved in this regulation, particularly those that function by direct interaction with DNA at the site of the break. To carry out this analysis, we have focused on the well-characterized DSB that occurs at the MAT locus during mating type switching.The MAT locus is the central element in the yeast mating type system (reviewed in reference 10). It exists in two allelic states, known as MATa and MAT␣, which determine whether a haploid cell has an a or ␣ mating type, respectively. MAT contains five sequence blocks: W, X, Y, Z1, and Z2 (1) (Fig. 1). The W, X, Z1, and Z2 blocks are identical in...
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