Genome stability in rad mutants

描述(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造成的DNA损伤的重要途径。然而，重组必须仔细控制，因为大量分散的重复序列之间的不受限制的重组可能会导致有害的基因组重组。由于在真核生物中，这些重复序列中的大多数都是300bp或更短的，所以对这些短序列之间的重组的控制比对长序列的控制更严格。已知的在萌芽酵母中介导辐射反应的几个基因也控制着短序列重组(SSR)。拟议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的因素之间的重要相互作用。在含有突变SSR因子的细胞中的DNA片段插入分析将被用来探索特定缺陷对SSR的影响，无论是单独的还是联合的。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。连接介导的聚合酶链式反应将被用来跟踪和定量在SSR过程中产生的次级断裂。染色质免疫沉淀将被用来跟踪几种重要的蛋白质复合体与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和维持基因组稳定性至关重要的几个分子相互作用的更彻底的了解。

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.