MECHANISM OF DNA RECOMBINATION AND REPAIR IN YEAST SACCHAROMYCES CEREVISIAE

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Proteins binding to single-stranded DNA are expected to participate in DNA replication, recombination and repair. Several single-stranded DNA binding proteins (SSBs) (14KD, 20KD, 26KD, 35KD, 38KD, 42KD and 45KD) have been purified from the yeast Saccharomyces cerevisiae and antibodies have been raised against them. Using the antibodies as probes, the SSB genes have been identified and sequenced. Deletions and/or disruptions of these genes have been constructed and the resulting phenotypes studied. At least the 20KD SSB gene is essential for cell viability. An ATP-independent activity that catalyzes the transfer of one strand from a duplex linear molecule of DNA to a complementary circular single strand has been detected in crude extracts from mitotic and meiotic cells by adding yeast SSBs. This activity increases more than 15-fold during meiosis in MATalpha/MATalpha diploids, but not in neither MATalpha/MATalpha or MATalpha/MATalpha. The polypeptide responsible for this activity was purified to homogeneity from meiotic cells and characterized. Although this yeast strand- exchange protein (ySEP) has properties similar to those of prokaryotic SEPs and some eucaryotic SEPs, it is distinct because it requires no nucleotide cofactor, its reaction is efficient and rapid, and catalytic amounts of the protein are required for the reaction. This meiotic ySEP activity is controlled by the RAD50 and RAD52 genes which are required for meiotic recombination as well as sporulation. Both monoclonal and monospecific polyclonal antibodies have been raised against the homogeneous ySEP and identification and cloning of the gene are underway.