Genome stability in rad mutants

描述（由申请人提供）：同源重组是修复辐射、化学物质和内源性细胞过程引起的DNA损伤的重要途径。然而，重组必须仔细控制，因为在众多分散的重复序列之间不受限制的重组可能导致有害的基因组重排。由于这些重复序列在真核生物中大多数是300 bp或更短，因此这些短序列之间的重组控制比长序列更严格。在出芽酵母中介导辐射反应的几个已知基因也控制短序列重组（SSR）。提出的研究计划的目标是在遗传和分子水平上进一步研究几个最关键因素之间的重要相互作用。在突变SSR因子的细胞中，DNA片段插入试验将用于探索特定缺陷（单独或联合）对SSR的影响。通过重组修复基因组双链断裂将用于监测这些突变体中SSR的动力学。结扎介导PCR将用于跟踪和定量SSR过程中产生的次级断裂。染色质免疫沉淀将用于跟踪几种重要蛋白质复合物和重组底物的相互作用。这种遗传和物理方法的结合将促进对SSR至关重要的几个分子相互作用的更深入的理解，并维持基因组的稳定性。

DESCRIPTION (provided by applicant): 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.