In prokaryotic systems, helicases have well defined roles in recombination. Dr. Hannah Klein previously identified mutants in yeast that have a mitotic hyper-rec phenotype for substrates with direct repeats. Two of these mutants, hpr5/srs2 and hpr4, are the subject of this proposal. Dr. Klein has shown that Srs2p is a DNA helicase. The protein has homology to E. coli uvrD, and like UvrD has 3' to 5' helicase activity. The results of epistasis analysis of the srs2 mutation with mutations affecting DNA repair (RAD6 epistasis group) or recombination (RAD52 epistasis group) have led Dr. Klein to propose that the hyper-rec phenotype of srs2 mutants results from channeling DNA lesions normally repaired by the error-prone repair pathway into a recombination pathway. A srs2 null mutant is viable, but shows synthetic lethality with rad54 and poor growth with rad50. She has made use of the synthetic lethal interaction with rad54 to identify temperature-sensitive alleles of srs2. In contrast to the hyper-rec mitotic phenotype exhibited by srs2-101, the mutant shows a mitotic hypo-rec phenotype, with both spore viability and recombination reduced about two-fold. In a search of the yeast genome for potential helicases, Dr. Klein identified an open reading frame with homology to RAD54 that she calls RDH54. srs2 rdh54 double mutants are viable as haploids, but show a synthetic lethal phenotype in diploids. She has identified a second open reading frame with significant homology to RAD54 that she plans to characterize. Dr. Klein has found that hpr4 mutants have some phenotypes similar to srs2 mutants, and that the two genes are probably in the same epistasis group. However, hpr4 mutants have a mutator phenotype not shared by srs2 mutants, and other phenotypes that suggest the Hpr4p may function in DNA mismatch repair. Dr. Klein proposes five specific aims. First, genetic interactions of srs2 with helicase homolog genes will be studied with the goal of understanding possible overlaps in function between Srs2p and the helicase homologs and at defining multiple recombination repair pathways. Second, proteins that interact with Srs2p will be sought with the goal of determining if Srs2p acts in a complex. Third, the Srs2p DNA helicase will be tested for its ability to promote in vitro branch migration of Holliday junctions. Fourth, strains carrying conditional alleles of srs2 will be examined to determine when during the mitotic and meiotic cell cycles Srs2p functions. Finally, the HPR4 gene will be cloned and null mutants constructed and characterized.
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