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H2AX控制双链断裂（DSB）细胞反应的机制，以及它如何在双链断裂修复（DSBR）中起作用。H2AX在其C端尾部139丝氨酸上发生磷酸化以响应DNA损伤。缺乏H2AX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基因，用于分析体细胞中主要的同源重组途径姐妹染色单体重组（SCR）。我们发现H2AX丝氨酸139控制HR，包括SCR。值得注意的是，H2AX的这一功能在整个进化过程中似乎是保守的。此外，我们发现H2AX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）。其他研究表明H2AX在DSBR的第三个主要通路non- homoous endjoining （NHEJ）中发挥作用。我们认为，除了丝氨酸139之外，H2AX的结构元素可能参与了H2AX的重组功能。为了验证这一假设，我们将定量评估H2AX单个残基在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H2AX磷酸化后被招募到染色质上。其中一些可能有助于h2ax依赖性重组功能。我们将通过识别新的H2AX相互作用伙伴，并通过研究这些相互作用伙伴和其他已知的H2AX相互作用伙伴在调节DSBR（包括HR/SCR、SSA和NHEJ）中的功能来验证这一假设。我们将尝试通过测量H2AX+/+与H2AX-/-等基因原代细胞中修复因子对DSB位点的招募来研究H2AX反应的动态方面（目的2）。重组缺陷经常导致其他基因的突变率增加。我们将评估H2AX功能障碍的致突变后果（目的3）。因此，这项工作将大大促进我们对H2AX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.