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突变和致癌至关重要。同源重组修复（Homologous recombinational repair， HRR）是酵母修复电离辐射（IR）诱导的双链断裂（DSBs）的主要途径，Rad51在其中起核心作用。然而，在哺乳动物细胞中，无论是生物化学机制还是HRR在细胞对DNA损伤反应中的相对贡献，都没有得到很好的理解。人类XRCC2蛋白与HRR有关，这是由于对ir敏感的XRCC2突变体irs1的特性，并发现它属于人类rad51样蛋白家族。此外，酵母2-杂交分析表明，XRCC2与另一个rad51样蛋白Rad51D存在物理相互作用。本项目将检验XRCC2通过与人类Rad51 （HsRad51）的功能关联参与HRR的假设。纯化后的XRCC2的生化活性，如atp酶，将被表征，并确认XRCC2与Rad51D的相互作用。需要解决的一个关键问题是XRCC2或XRCC2- rad51d复合物是否作为辅助因子促进HsRad51的链交换活性。XRCC2的atp酶及其与Rad51D的相互作用的生物学相关性将通过在仓鼠irs1细胞中表达突变cdna来评估。通过鉴定人类提取物中与XRCC2相互作用的其他蛋白，不仅可以了解Rad51家族是否存在于多蛋白复合物中，还可以分离出与XRCC2相互作用的新蛋白。XRCC2在ir诱导的HsRad51核病灶形成中的需求将在irs1细胞中被表征，XRCC2与Rad51的细胞共定位将被检查，以确定XRCC2与HsRad51的功能关联。最后，XRCC2的细胞周期依赖性表达和同步irs1细胞的放射敏感性将被确定，以测量XRCC2对HRR的定量贡献，HRR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XRCC2在哺乳动物HRR中的作用，以及该途径在维持染色体稳定性中的重要性。

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.