The major goal of this study is a detailed understanding of the molecular mechanisms of homologous recombination stimulated by double-strand breaks in DNA. Homologous recombination is an essential process in humans and all other eucaryotic organisms. Homothallic switching of mating-type genes in Saccharomyces cerevisiae occurs by homologous recombination (gene conversion) after the HO endonuclease creates a site-specific double-strand cleavage at the mating-type (MAT). Analysis of DNA extracted from cells in which switching is induced synchronously allows the physical analysis of the molecular events in this process. Intermediates of recombination are identified by gel electrophoresis or by sensitive polymerase chain reaction methods. The role(s) of various gene products important for DNA replication, repair and recombination are assessed by physical analysis of DNA from cells carrying mutations in these genes. In addition, a model plasmid recombination system is used to assess the properties of HO-induced recombination between homologous regions lacking the special constraints of MAT sequences. Experiments are designed to characterize more extensively two distinct pathways to repair double-strand breaks: a gap-filling mechanism leading to gene conversion events and a single-strand annealing mechanism that results exclusively in deletions between flanking homologous sequences. Finally, a comparison of events initiated by HO endonuclease and by the mitochondrial omega endonuclease will be conducted, to determine whether HO is involved in any other steps in recombination other than the creation of a double-strand break. A second focus of this proposal is the analysis of donor preference, whereby MATalpha cells interact preferentially with HMR and MATalpha cells choose HML. Both cis- and trans-acting mutations that alter the ability of MAT to pair efficiently with a donor, located nearly 200 kb away, are being analyzed. The possible role of these sequences in constraining crossing- over during switching will also be assessed. The way in which these distant sequences can be brought together for recombination may shed light on similar events in the generation of human immunoglobulin gene rearrangements.
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