Identification of a Protein That Binds to the Ho Endonuclease Recognition Sequence at the Yeast Mating Type Locus

Abstract

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...

Persistence of an alternate chromatin structure at silenced loci in the absence of silencers

“…Regulated cleavage of the site at MAT by the endogenous Ho endonuclease initiates matingtype interconversion (17). A Y͞Z junction binding protein, YZbp, was recently purified from yeast (30), and it was proposed that the factor facilitates cleavage by maintaining an open chromatin conformation (27,30). The loss of one supercoil at HMR upon derepression is consistent with YZbp, or some other factor, occluding or unwrapping nucleosomes (Fig.…”

Inteins: Structure, Function, and Evolution

“…The role of this endonuclease is to initiate a gene conversion event that results in a switch of the mating type. The HO endonuclease catalyzes a double-strand break in the MAT locus, which initiates recombination with one of two unlinked loci that interconvert MATa and MAT-α (53,101). The HO endonuclease is homologous to the homing endonucleases of the LAGLIDADG-type (17,69); in particular, the HO endonuclease is similar to the intein in the yeast vma-1 gene.…”

Investigation of the Mechanism of Meiotic DNA Cleavage by VMA1 -Derived Endonuclease Uncovers a Meiotic Alteration in Chromatin Structure around the Target Site

“…Such a coactivating mechanism can be found in the case of HO endonuclease in budding yeast. An activity to bind to a sequence near the target site of HO endonuclease was detected previously, and it is suggested that the formation of HO-mediated DSB in vivo is regulated by this coactivating sequence (38).…”