ROLE OF HISTONE H2AX IN DOUBLE STRAND BREAK REPAIR

描述（由申请人提供）：本提案的长期目标是确定组蛋白H2 AX控制细胞对双链断裂（DSB）的反应的机制以及它在双链断裂修复（DSBR）中的功能。H2 AX在其C末端尾的丝氨酸139上发生磷酸化以响应DNA损伤。缺乏H2 AX的小鼠表现出基因组不稳定性和癌症易感性。我们最近开发了一种新的报告分析姐妹染色单体重组（SCR），一个主要的同源重组（HR）途径在体细胞。我们发现H2 AX丝氨酸139控制HR，包括SCR。值得注意的是，H2 AX的这种功能似乎在进化过程中是保守的。此外，我们发现H2 AX调节不同DSBR途径之间的“选择”，有利于姐妹染色单体重组（SCR）和抑制单链退火（SSA）。其他工作表明H2 AX在第三个主要DSBR途径，非同源末端连接（NHEJ）中的作用。我们认为，除了丝氨酸139之外，H2 AX的结构元件可能有助于H2 AX重组功能。为了检验这一假设，我们将定量评估H2 AX的各个残基在HR/SCR、SSA和NHEJ中的作用（目的1）。许多DNA损伤响应蛋白复合物在H2 AX磷酸化后被募集到染色质。其中一些可能有助于H2 AX依赖性重组功能。我们将通过鉴定新的H2 AX相互作用伙伴并通过研究这些和其他已知的H2 AX相互作用物在DSBR调节中的功能（包括HR/SCR、SSA和NHEJ）来测试这一假设。我们将尝试通过测量修复因子在H2 AX +/+与H2 AX-/-等基因原代细胞中DSB位点的募集来检查H2 AX应答的动态方面（目的2）。重组中的缺陷经常导致其他基因突变率的增加。我们将评估H2 AX功能障碍的致突变后果（目的3）。因此，这项工作将大大推进我们对H2 AX如何作为肿瘤抑制基因的理解。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to determine the mechanisms by which histone H2AX controls cellular responses to double strand breaks (DSB) and how it functions in double strand break repair (DSBR). H2AX undergoes phosphorylation on serine 139 of its C terminal tail in response to DNA damage. Mice lacking H2AX exhibit genomic instability and cancer predisposition. We recently developed a novel reporter for analysis of sister chromatid recombination (SCR), a major homologous recombination (HR) pathway in somatic cells. We found that H2AX serine 139 controls HR, including SCR. Remarkably, this function of H2AX appears to be conserved across evolution. Further, we found that H2AX regulates the "choice" between distinct DSBR pathways, favoring sister chromatid recombination (SCR) and suppressing single strand annealing (SSA). Other work suggests a role for H2AX in the third major DSBR pathway, non-homolgous endjoining (NHEJ). We believe that structural elements of H2AX in addition to serine 139 likely contribute to H2AX recombination functions. To test this hypothesis, we will assess quantitatively the role of individual residues of H2AX in HR/SCR, SSA and NHEJ (Aim 1). A number of DNA damage responsive protein complexes are recruited to chromatin following H2AX phosphorylation. Some of these may contribute to H2AX-dependent recombination functions. We will test this hypothesis by identifying new H2AX interaction partners and by studying the function of these and other known H2AX interactors in regulation of DSBR, including HR/SCR, SSA and NHEJ. We will attempt to examine dynamic aspects of the H2AX response by measuring the recruitment of repair factors to the site of a DSB in H2AX+/+ vs. H2AX-/- isogenic primary cells (Aim 2). Defects in recombination frequently cause increased mutation rates in other genes. We will assess the mutagenic consequences of H2AX dysfunction (Aim 3). This work will therefore significantly advance our understanding of how H2AX acts as a tumor suppressor gene.