The Fanconi Anemia Core and D2 Complexes

Fanconi 贫血核心和 D2 复合物

• 批准号: 7065242
• 负责人: GARY M KUPFER
• 金额: $ 37.06万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7065242
• 关键词: DNA damage; HeLa cells; RNA binding protein; RNA interference; SDS polyacrylamide gel electrophoresis; binding proteins; cell cycle; chromatin; congenital aplastic anemia; gel mobility shift assay; gene complementation; immunoprecipitation; mass spectrometry; nuclear matrix; phosphorylation; polymerase chain reaction; posttranslational modifications; protein localization; protein purification; protein structure function; western blottings

DESCRIPTION (provided by applicant): The study of rare genetic disorders of cancer susceptibility, such as Fanconi anemia (FA) have led to seminal advances in cancer biology as a whole. The hallmark of FA is genomic instability and DNA damage hypersensitivity, but the normal function of the FA pathway or mechanism leading to cancer in the affected patients remains unknown. FA is accounted for by multiple complementation groups corresponding to at least 8 cloned genes whose encoded proteins contain few functional motifs. What is known is that the FA pathway interacts with other more defined pathways involved in cellular response to DNA damage, such as ATM and BRCA1. The Kupfer laboratory has established that FA proteins such as FANCA and FANCG phosphorylated, that the FA core complex (composed of at least 5 FA proteins) localizes to at least 3 subcellular compartments, including chromatin, and that the core complex binds to additional non-characterized proteins. This proposal centers on the analysis of posttranslational modifications of the FA proteins FANCA and FANCG and on detecting new binding proteins and determining their function. This would shed light on the long-term goal of the Kupfer laboratory: define the mechanisms of genomic instability caused by FA as well as understand the normal function of the FA proteins. In this proposal the experiments proposed address the central hypothesis: that the phosphorylation of FANCA and FANCG regulate proper localization to chromatin and that a novel FA binding protein, RBM10, has RNA binding activity critical for this localization. The work proposed will 1) determine the cause and effect of FANCA and FANCG phosphorylation, 2) analyze RBM10 and RNA function in the FA core complex, and 3) focus on purifying additional FA core complex and FANCD2 binding proteins, especially from chromatin. This research is significant because it sheds light on a mechanism of genomic instability at the level of the functional genetic unit of the cell: chromatin. Because the response to DNA damage involves numerous intersecting pathways, defining the function of FA proteins will allow us to link these disparate pathways in a mechanism explaining cancer formation.

描述（由申请人提供）：对癌症易感性的罕见遗传性疾病（例如范可尼贫血（FA））的研究导致了整个癌症生物学的开创性进展。 FA 的标志是基因组不稳定和 DNA 损伤超敏反应，但 FA 途径的正常功能或导致受影响患者癌症的机制仍不清楚。 FA 由多个互补组负责，这些互补组对应于至少 8 个克隆基因，其编码的蛋白质含有很少的功能基序。众所周知，FA 途径与参与细胞对 DNA 损伤反应的其他更明确的途径（例如 ATM 和 BRCA1）相互作用。 Kupfer 实验室已确定 FA 蛋白（如 FANCA 和 FANCG）磷酸化，FA 核心复合物（由至少 5 个 FA 蛋白组成）定位于至少 3 个亚细胞区室，包括染色质，并且核心复合物与其他非特征蛋白质。该提案的重点是分析 FA 蛋白 FANCA 和 FANCG 的翻译后修饰，以及检测新的结合蛋白并确定其功能。这将阐明 Kupfer 实验室的长期目标：定义 FA 引起的基因组不稳定机制并了解 FA 蛋白的正常功能。在该提案中，提出的实验提出了中心假设：FANCA 和 FANCG 的磷酸化调节染色质的正确定位，并且新型 FA 结合蛋白 RBM10 具有对此定位至关重要的 RNA 结合活性。拟议的工作将 1) 确定 FANCA 和 FANCG 磷酸化的因果关系，2) 分析 FA 核心复合物中的 RBM10 和 RNA 功能，3) 重点纯化其他 FA 核心复合物和 FANCD2 结合蛋白，特别是从染色质中纯化。这项研究意义重大，因为它揭示了细胞功能遗传单元（染色质）水平上基因组不稳定的机制。由于对 DNA 损伤的反应涉及许多交叉途径，因此定义 FA 蛋白的功能将使我们能够将这些不同的途径联系起来，以解释癌症形成的机制。