Biochemical Analysis of the BRCA1 Protein Complex

描述（由申请人提供）：该竞争性延续拨款（CA84199）的长期目标是了解肿瘤抑制因子BRCA1蛋白的功能。我们之前纯化并鉴定了BRCA1蛋白复合物BASC。该复合体的组成使我们提出BASC作为基因组监视复合体的功能，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。我们将进一步验证基因组监视复合体假说。尽管越来越多的证据表明BRCA1在DNA损伤反应中起作用，但BRCA1及其相关伙伴在DNA损伤反应概念框架中的确切作用需要解决。我们假设TopBP1作为适配器起作用，并与BRCA1形成一个检查点模块，以响应S. cerevisiae中的scRad9和scRad53的DNA损伤。因此，这一假设扩大了效应酶的活性，将DNA损伤反应中的E3连接酶包括在内，增加了迄今为止激酶的效应酶（Chkl和Chk2）。此外，我们认为BRCA1通过其底物发挥其功能。BRCA1的许多相关功能通常看起来不相关且令人困惑，现在可以将其合理化为不同底物的作用。鉴定执行检查点激活的BRCA1底物是本提案的另一个目标。具体目的是：(1)验证BASC中的RFC和/或BLM在DNA复制应激反应中上游发挥作用的假设，(2)在DNA损伤后纯化BRCA1复合物，(3)验证TopBP1和BRCA1形成与scRad9和scRad53相似的检查点模块的假设，以及(4)鉴定和表征BRCA1泛素连接酶活性底物。

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.