Chromatin Regulation of Double-Strand Break Repair

描述(由申请人提供)：DNA修复和DNA损伤检查点对肿瘤抑制很重要，是肿瘤对放射和化疗耐药的关键决定因素。DNA双链断裂(DSB)是由遗传毒性化学物质和电离辐射产生的，在DNA复制过程中自发发生。双链断裂通过非同源末端连接(NHEJ)和同源重组(HR)修复。双链断裂发生在染色质的背景下，染色质的改变在DNA修复和检查点蛋白到损伤部位的募集中起着重要的作用。本研究旨在确定染色质修饰和重塑在DSB蛋白募集中的作用，以及在酵母中DSB修复结果的调节。我们的中心假设是DSB修复效率和结果是通过修复蛋白、检查点蛋白和染色质动力学的综合作用来调节的。我们将通过追求三个特定的目标来验证这一假设，这些目标集中在(1)染色质变化在向DSB募集修复和检查点蛋白中的作用；(2)确定在HR过程中DSB位点和供体位置的染色质变化；以及(3)确定染色质如何调节DSB修复。与组蛋白H2A的磷酸化一样，我们发现核小体置换在修复蛋白及时募集到DSB中发挥了作用。这些和其他染色质改变在蛋白质募集到DSB、调节DSB修复和检查点激活中的作用将被调查。这些研究将阐明染色质改变如何调节细胞对DSB的反应，从而确定DSB损伤的遗传后果。拟议的研究是相关的，因为染色质修饰、DSB修复和检查点反应在酵母和人类之间是保守的。这些研究意义重大，因为它们将揭示DSB修复的新调控机制，从而确定可用于癌症放射治疗和化疗的新靶点。

DESCRIPTION (provided by applicant): DMA repair and DNA damage checkpoints are important for tumor suppression, and are critical determinants of tumor resistance to radiation and chemotherapeutics. DNA double-strand breaks (DSBs) are produced by genotoxic chemicals and ionizing radiation, and arise spontaneously during DNA replication. DSBs are repaired by non-homologous end-joining (NHEJ) and homologous recombination (HR). DSBs occur in the context of chromatin, and chromatin alterations play important roles in the recruitment of DNA repair and checkpoint proteins to damage sites. The proposed studies are designed to determine the roles of chromatin modification and remodeling in protein recruitment to DSBs, and in the regulation of DSB repair outcome in the yeast Saccharomyces cerevisiae. Our central hypothesis is that DSB repair efficiency and outcome are regulated through the integrated actions of repair proteins, checkpoint proteins, and chromatin dynamics. We will test this hypothesis by pursuing three Specific Aims focused on (1) the roles of chromatin changes in recruitment of repair and checkpoint proteins to DSBs; (2) defining chromatin changes at a DSB site and a donor locus during HR; and (3) determining how chromatin regulates DSB repair. As with phosphorylation of histone H2A, we found that nucleosome displacement plays a role in the timely recruitment of repair proteins to DSBs. The roles of these and other chromatin alterations in protein recruitment to DSBs, regulation of DSB repair, and checkpoint activation will be investigated. These studies will clarify how chromatin alterations regulate cellular responses to DSBs and thereby determine the genetic consequences of DSB damage. The proposed studies are relevant because chromatin modifications, DSB repair, and checkpoint responses are conserved from yeast to man. The studies are significant because they will reveal new regulatory mechanisms in DSB repair, and thus identify new targets to exploit in cancer radio- and chemotherapy.