Mammalian Replication fork stalling

描述（由申请人提供）：本提案的目的是开发新的工具，用于研究哺乳动物细胞中停滞或断裂的复制叉与姐妹染色单体重组之间的关系。尽管目前的模型提出，在停滞的DNA合成位点的重组对于防止基因组不稳定性和癌症是至关重要的，但在哺乳动物细胞中模拟该过程的能力受到严重限制，这是由于缺乏可以以定量和机械方式准确模拟该过程的重组报告基因。本文提出的工作将提供这样的工具，通过将同源重组和姐妹染色单体重组的“最先进的”哺乳动物报告基因与引起位点特异性复制叉停滞的新策略相结合。这项工作将提供一个非常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.调整已知的复制叉屏障，用于在哺乳动物细胞中诱导位点特异性HR。2.研究哺乳动物细胞中复制叉屏障处的HR和SCR。3.开发I-SceI的“切口酶”突变体，以刺激哺乳动物细胞中染色体I-SceI位点的HR和SCR。 公共卫生相关性：通过称为“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过早衰老和癌症至关重要。当DNA复制机器在受损DNA上停顿时，SCR被触发;然而，没有工具可用于研究哺乳动物细胞中复制停滞和SCR之间的关系。该提案将解决这一需求，通过开发两个新的系统，激发复制停滞在一个特定的染色体位点在活的哺乳动物细胞，并通过测量刺激SCR在这些事件的反应。

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.