Biochemical Analysis of the BRCA1 Protein Complex

描述(由申请人提供)：这项竞争性继续授予(CA84199)的长期目标是了解肿瘤抑制因子BRCA1蛋白的功能。我们先前纯化并鉴定了BRCA1蛋白复合体BASC。这种复合体的组成使我们提出BASC作为基因组监测复合体的功能，其中DNA修复蛋白在DNA损伤反应途径的上游发挥作用，以检测不同类型的DNA损伤。我们将进一步检验基因组监测复合体假说。尽管越来越多的证据表明BRCA1在DNA损伤反应中发挥作用，但BRCA1及其相关伙伴在DNA损伤反应概念框架中的确切作用仍有待研究。我们假设TopBP1作为一个接头，与BRCA1形成一个检查点模块，以应对酿酒酵母中与scRad9和scRad53类似的DNA损伤。因此，这一假说扩展了效应器的酶活性，将E3连接酶包括在DNA损伤反应中，增加了迄今为止的激酶(Chk1和Chk2)的效应酶。此外，我们认为BRCA1通过其底物发挥其功能。BRCA1的许多看似无关和令人困惑的隐含功能现在可能被合理化为不同底物的影响。确定BRCA1底物以执行检查点激活是本提案的另一个目标。其具体目的是(1)检验BASC内RFC和/或BLM在DNA复制应激反应中上游功能的假说，(2)DNA损伤后纯化BRCA1复合体，(3)检验TopBP1和BRCA1形成与scRad9和scRad53类似的检查点模块的假说，以及(4)鉴定和表征BRCA1泛素连接酶活性的底物。

DESCRIPTION (provided by applicant): The long-term goal of this competing continuation of grant (CA84199) is to understand the functions of tumor suppressor BRCA1 protein. We previously purified and identified a BRCA1 protein complex, BASC. The composition of this complex has led us to propose that BASC functions as genome surveillance complex in which the DNA repair proteins function in the upstream of the DNA damage response pathway to detect DNA lesions of different types. We will further test the genome surveillance complex hypothesis. Despite the mounting evidence that BRCA1 functions in DNA damage response, the precise roles of BRCA1 and its associated partners in the conceptual framework of DNA damage response need to be addressed. We hypothesize that TopBP1 functions as an adaptor and forms a checkpoint module with BRCA1 in response to DNA damage that parallels that of scRad9 and scRad53 in S. cerevisiae. This hypothesis thus expands the effector enzymatic activity to include an E3 ligase in the DNA damage response, adding to the effector enzymes of kinases so far (Chkl and Chk2). Furthermore, we propose that BRCA1 exerts its functions through its substrates. The many implicated functions of BRCA1 that are often seemingly unrelated and confusing may now be rationalized as the effects of different substrates. The identification of BRCA1 substrates through which the checkpoint activation is executed is another goal of this proposal. The specific aims are (1) To test the hypothesis that RFC and/or BLM within the BASC function upstream in the response to DNA replication stress, (2) To purify BRCA1 complexes after DNA damage, (3) To test the hypothesis that TopBP1 and BRCA1 form a checkpoint module that parallels that of scRad9 and scRad53, and (4) To identify and characterize substrates of the BRCA1 ubiquitin ligase activity.