Genome stability in rad mutants
Homologous recombination is an important pathway for the repair of DNA damage by radiation, chemicals, and endogenous cellular processes. However, recombination must be carefully controlled as deleterious genome rearrangements can result from unrestricted recombination between numerous, dispersed repetitive sequences. Since the majority of these repeats are 300 bp or shorter in eukaryotes, the control of recombination between these short sequences is more stringent than for longer sequences. Several of the genes known to mediate the response to radiation in the budding yeast S. cerevisiae also control short-sequence recombination (SSR). The goal of the proposed research program is to further investigate important interactions between several of the most critical factors at the genetic and molecular levels. DNA fragment insertion assays in cells with mutant SSR factors will be used to explore the effect of specific defects, both alone and in combination, on SSR. Repair of a genomic double-strand break by recombination will be used to monitor the kinetics of SSR in these mutants. Ligation mediated PCR will be employed to track and quantitate secondary breaks created during SSR. Chromatin immunoprecipitation will be used to follow the interaction of several important protein complexes and the recombination substrates. This combination of genetic and physical methods will promote a more thorough understanding of several of the molecular interactions critical for SSR, and the maintenance of genome stability.