Biochemical Analysis of the BRCA1 Protein Complex

BRCA1 蛋白复合物的生化分析

• 批准号: 6680632
• 负责人: JUN QIN
• 金额: $ 28.29万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-12-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6680632
• 关键词: DNA damage; DNA repair; DNA replication; HeLa cells; SDS polyacrylamide gel electrophoresis; brca gene; enzyme activity; genetic transcription; high performance liquid chromatography; immunoprecipitation; ligase; mass spectrometry; molecular oncology; neoplasm /cancer genetics; phosphorylation; protein protein interaction; protein purification; protein structure function; tissue /cell culture; tumor suppressor proteins; western blottings

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.

描述（由申请人提供）：这项竞争性延续资助 (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发挥作用，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需解决。我们假设 TopBP1 作为适配器发挥作用，并与 BRCA1 形成检查点模块，以响应 DNA 损伤，这与酿酒酵母中的 scRad9 和 scRad53 类似。因此，该假设扩展了效应酶活性以将E3连接酶包括在DNA损伤反应中，从而添加到迄今为止的激酶的效应酶（Chk1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似无关且令人困惑，现在可能被合理化为不同底物的影响。鉴定执行检查点激活的 BRCA1 底物是该提案的另一个目标。具体目标是 (1) 检验 BASC 内的 RFC 和/或 BLM 在 DNA 复制应激反应中发挥上游功能的假设，(2) 在 DNA 损伤后纯化 BRCA1 复合物，(3) 检验 TopBP1 和 BRCA1 形成与 scRad9 和 scRad53 平行的检查点模块的假设，以及 (4) 识别和表征 BRCA1 的底物 泛素连接酶活性。