BIOLOGICAL FUNCTION OF XRCC2 IN RECOMBINATIONAL REPAIR

DNA修复过程对于维持基因组完整性和防止诱变和致癌是必不可少的。同源重组修复（HRR）是电离辐射（IR）诱导的酵母双链断裂（DSB）修复的主要途径，其中Rad 51起着核心作用。然而，在哺乳动物细胞中，无论是生化机制，也没有相对贡献的HRR细胞响应DNA损伤是很好的理解。 通过IR敏感性XRCC 2突变体irs 1的特性和属于Rad 51样蛋白的人类家族的发现，人类XRCC 2蛋白与HRR有关。 此外，酵母双杂交分析表明XRCC 2与另一种Rad 51样蛋白Rad 51 D的物理相互作用。 该项目将测试XRCC 2通过与人Rad 51（HsRad 51）的功能关联参与HRR的假设。 将表征纯化的XRCC 2的生化活性，如ATP酶，并将证实XRCC 2与Rad 51 D的相互作用。 需要解决的关键问题是XRCC 2或XRCC 2-Rad 51 D复合物是否作为辅助因子促进HsRad 51的链交换活性。通过在仓鼠irs 1细胞中表达突变cDNA以检测互补作用，评价XRCC 2 ATP酶的生物学相关性以及与Rad 51 D的相互作用。 通过鉴定人提取物中与XRCC 2相互作用的其他蛋白质，不仅可以了解Rad 51家族是否存在于多蛋白复合物中，还可以分离与XRCC 2相互作用的新蛋白质。 IR诱导的HsRad 51核灶形成中对XRCC 2的需求将在irs 1细胞中表征，并且将检查XRCC 2与Rad 51的细胞共定位以确定XRCC 2与HsRad 51的功能关联。 最后，将确定XRCC 2的细胞周期依赖性表达和同步化irs 1细胞的放射敏感性，以测量XRCC 2对HRR的定量贡献，其可能仅在细胞周期的S和G2期起作用。 这项研究将有助于更好地了解XRCC 2在哺乳动物HRR中的作用以及该途径在维持染色体稳定性方面的重要性。

DNA repair processes are essential for maintaining genome integrity and preventing mutagenesis and carcinogenesis. Homologous recombinational repair (HRR), in which Rad51 plays a central role, is a major pathway in yeast for repairing double strand breaks (DSBs) induced by ionizing radiation (IR). However, in mammalian cells neither the biochemical mechanism nor the relative contribution of HRR in cellular response to DNA damage is well understood. The human XRCC2 protein is implicated in HRR by the properties of the IR-sensitive XRCC2 mutant irs1 and the finding that is belongs to a human family of Rad51-like proteins. Also, physical interaction of XRCC2 with another Rad51-like protein, Rad51D, is suggested by yeast 2-hybrid analysis. This project will test the hypothesis that XRCC2 participates in HRR through functional association with human Rad51 (HsRad51). Biochemical activities, such as ATPase, of purified XRCC2 will be characterized, and the interaction of XRCC2 with Rad51D will be confirmed. A key question to be addressed is whether XRCC2 or XRCC2-Rad51D complex acts as an accessory factor to promote HsRad51's strand exchange activity. The biological relevance of XRCC2's ATPase and interaction with Rad51D will be evaluated by expressing mutant cDNAs in hamster irs1 cells to test for complementation. By identifying the other proteins that interact with XRCC2 in human extracts, an attempt will made not only to learn whether the Rad51 family is present in a multiprotein complex, but also to isolate novel proteins interacting with XRCC2. The requirement for XRCC2 in IR-induced HsRad51 nuclear focus formation will be characterized in irs1 cells and cellular colocalization of XRCC2 with Rad51 will be examined to define a functional association of XRCC2 with HsRad51. Finally, cell cycle-dependent expression of XRCC2 and the radiosensitivity of synchronized irs1 cells will be determined to measure the quantitative contribution of XRCC2 to HRR, which likely operates only in the S and G2 phases of the cell cylce. This study will lead to a better understanding of the role of XRCC2 in mammalian HRR and the importance of this pathway in maintaining chromosomal stability.