ROLE OF HISTONE H2AX IN DOUBLE STRAND BREAK REPAIR

描述(由申请人提供)：这项建议的长期目标是确定组蛋白H2AX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)中的作用。作为对DNA损伤的反应，H2AX在其C末端尾部的丝氨酸139上经历了磷酸化。缺乏H2AX的小鼠表现出基因组不稳定和癌症易感性。我们最近开发了一种新的报告程序来分析姐妹染色单体重组(SCR)，这是体细胞中一种主要的同源重组(HR)途径。我们发现，H_2AX丝氨酸139控制心率，包括Scr。值得注意的是，H2 AX的这一功能似乎在进化过程中是保守的。此外，我们发现H_2AX调节着不同的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，H2AX在第三个主要的DSBR途径，非同源末端连接(NHEJ)中起作用。我们认为，除了丝氨酸139外，H_2AX的结构元件可能参与了H_2AX的重组功能。为了验证这一假设，我们将定量评估H2 AX的单个残留物在HR/SCR、SSA和NHEJ(目标1)中的作用。在H_2AX磷酸化后，许多DNA损伤反应蛋白复合体被招募到染色质。其中一些可能有助于H_2AX依赖的重组功能。我们将通过寻找新的H_2AX相互作用伙伴，并研究这些和其他已知的H_2AX相互作用因子在调节DSBR中的功能，包括HR/SCR、SSA和NHEJ来验证这一假说。我们将试图通过测量修复因子在H_2AX+/+与H_2AX-/-等基因原代细胞中DSB位置的募集来检测H_2AX反应的动态方面(目标2)。重组缺陷经常会导致其他基因的突变率增加。我们将评估H_2AX功能障碍的诱变后果(目标3)。因此，这项工作将极大地促进我们对H2 AX作为肿瘤抑制基因的作用的理解。

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.