The Fanconi Anemia Core and D2 Complexes

描述（由申请人提供）：对罕见的癌症易感性遗传疾病的研究，如范可尼贫血（FA），已经导致了癌症生物学的重大进展。FA的标志是基因组不稳定和DNA损伤过敏，但FA途径的正常功能或导致癌症的机制在受影响的患者中仍不清楚。FA由至少8个克隆基因对应的多个互补组来解释，这些克隆基因编码的蛋白质含有很少的功能基序。已知的是，FA通路与其他更明确的参与细胞对DNA损伤反应的通路相互作用，如ATM和BRCA1。Kupfer实验室已经确定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，并且核心复合物结合其他非特征蛋白。本课题主要分析FA蛋白FANCA和FANCG的翻译后修饰，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：定义由FA引起的基因组不稳定的机制，并了解FA蛋白的正常功能。在这个提案中，实验提出了解决中心假设：FANCA和FANCG的磷酸化调节染色质的适当定位，并且一种新的FA结合蛋白RBM10具有对这种定位至关重要的RNA结合活性。我们的工作将包括：1)确定FANCA和FANCG磷酸化的原因和影响；2)分析RBM10和RNA在FA核心复合体中的功能；3)重点纯化FA核心复合体和FANCD2结合蛋白，特别是从染色质中纯化。这项研究意义重大，因为它揭示了细胞的功能遗传单位：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的途径，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。

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.