Mammalian Replication fork stalling

描述(申请人提供)：这项建议的目的是开发新的工具来研究哺乳动物细胞中停滞或断裂的复制叉子与姐妹染色单体重组之间的关系。尽管目前的模型表明，停滞的DNA合成位点上的重组对于预防基因组不稳定和癌症至关重要，但在哺乳动物细胞中模拟这一过程的能力受到严重限制，因为缺乏能够以定量和机械的方式准确模拟这一过程的重组记者。本文提出的工作将提供这样的工具，将同源重组和姐妹染色单体重组的“最新”哺乳动物记者与导致位点特异性复制分叉停滞的新策略相结合。这项工作将提供一个迫切需要的新工具来研究这一对人类癌症易感性至关重要的领域。我们的具体目标是：1.使已知的复制叉状屏障在哺乳动物细胞中诱导位置特异性HR。2.研究哺乳动物细胞中复制分叉障碍处的HR和SCR。3.发展I-SCEI的“镍酶”突变体，在哺乳动物细胞的染色体I-SCEI位点刺激HR和SCR。 公共卫生相关性：通过一种称为“姐妹染色单体重组”(SCR)的过程准确修复染色体断裂对于预防早衰和癌症至关重要。当DNA复制机器在受损的DNA上停止工作时，就会触发SCR；然而，目前还没有工具来研究哺乳动物细胞中复制停止和SCR的关系。这项建议将解决这一需求，通过开发两个新的系统，在活的哺乳动物细胞中的特定染色体位置引发复制停止，并通过测量SCR对这些事件的刺激。

DESCRIPTION (provided by applicant): The aim of this proposal is to develop novel tools for studying the relationship between stalled or broken replication forks and sister chromatid recombination in mammalian cells. Although current models propose that recombination at sites of stalled DNA synthesis is critical for preventing genomic instability and cancer, the ability to model this process in mammalian cells is severely limited, due to the absence of recombination reporters that can accurately model this process in a quantitative and mechanistic fashion. Work proposed herein will provide such tools, by combining "state-of-the-art" mammalian reporters of homologous recombination and sister chromatid recombination with novel strategies to cause site-specific replication fork stalling. This work will provide a much needed new tool to study this area of critical importance for human cancer predisposition. Our specific aims are: 1. Adapt a known replication fork barrier for the induction of site-specific HR in mammalian cells. 2. Study HR and SCR at a replication fork barrier in mammalian cells. 3. Develop "nickase" mutants of I-SceI to stimulate HR and SCR at a chromosomal I-SceI site in mammalian cells. PUBLIC HEALTH RELEVANCE: The accurate repair of chromosome breaks by a process termed "sister chromatid recombination" (SCR) is critical for preventing premature aging and cancer. SCR is triggered when the DNA replication machine stalls on damaged DNA; however, no tools are available to study the relationship between replication arrest and SCR in mammalian cells. This proposal will address this need, by developing two new systems for provoking replication arrest at a specific chromosome site in living mammalian cells, and by measuring the stimulation of SCR in response to these events.