Structure and Function of the Tumor Suppressor Protein BRCA2

肿瘤抑制蛋白 BRCA2 的结构和功能

基本信息

批准号：
9014530
负责人：
Jie Liu
金额：
$ 17.06万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-03-01 至 2018-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9014530
关键词：
Achievement Antibodies Architecture BRCA2 Protein BRCA2 gene Binding Biochemical Biochemistry Biological Markers C-terminal Centrifugation Clinical Research Collection Complex Critical Pathways DNA DNA Binding DNA Damage DNA Double Strand Break DNA Repair DNA Repair Pathway DNA-Binding Proteins Data Defect Dissociation Drug Targeting Electron Microscopy Equilibrium Filament Floor Future Genetic Recombination Genomic Instability Genotoxic Stress Goals Health Human Image Image Analysis Knowledge Lead Length Licensing Malignant Neoplasms Mediating Modeling Molecular Molecular Sieve Chromatography Morphologic artifacts Mutation Nature Negative Staining Pathway interactions Phenocopy Predisposition Preparation Process Property Proteins Resolution Resources Route Sampling Seminal Shapes Side Signal Transduction Staining method Stains Structure Sucrose System Testing Tumor Suppressor Proteins Work crosslink dimer homologous recombination image reconstruction improved in vivo monomer particle reconstruction repaired scaffold three dimensional structure tool

项目摘要

DESCRIPTION (provided by applicant): The tumor suppressor BRCA2 is a critical factor in homologous recombination (HR)-dependent DNA repair and disabling mutations lead to increased genome instability and cancer predisposition. The essential function of BRCA2 is to facilitate nucleation of the RAD51-ssDNA filament, a rate-limiting step for the formation of this central HR intermediate. BRCA2 engages in interactions with multiple proteins including post-translational modifiers and other regulators. The long-term goal is to elucidate the mechanism of BRCA2 and its binding partners in HR. This project focuses on one specific aim, the overall structure and architecture of full-length human BRCA2 and its modulation by substrate and binding partners. This Specific Aim is: Specific Aim 1: Structure and function of full-length human BRCA2 protein. DSS1 is a critical BRCA2 interaction partner. DSS1 depletion and DSS1 mutations phenocopy a BRCA2 defect. However, the precise mechanism by which DSS1 functions in BRCA2-mediated HR remains to be determined. Our preliminary data suggest an effect of DSS1 on BRCA2 architecture, and thus DSS1 biochemistry is incorporated as Subaim 1A to integrate functional studies to the structural approach. We will test the hypothesis that DSS1 stabilizes monomeric BRCA2 on ssDNA to nucleate RAD51 filament formation. The possibility of obtaining a crystal structure of full-length human BRCA2 is not feasible at present because of the size and the segmental nature of the protein. We will use TEM and cryo-EM to determine the overall structure of BRCA2 alone and in functional complexes with DNA, DSS1, and RAD51, to elucidate the effects of its substrate and main binding partners on the overall architecture of BRCA2. Preliminary data show that we can visualize full-length human BRCA2, whose identity was confirmed by immunogold-antibody staining and targeting the C-terminal His tag. Upon binding DSS1 and ssDNA, BRCA2 transitions to an open C-shape monomer with a significant conformational change from the monomers or dimers found in solution. Using the extraordinary EM resources of our collaborator Dr. Al-Bassam, we will reconstruct 3D structures of human BRCA2 using TEM imaging (Subaim 1B) and cryo-EM (Subaim 1C). We are fully aware of image reconstruction artifacts, and we safeguard against this by having two independent reconstruction efforts by Drs. Al-Bassam and Stahlberg.

描述（由申请人提供）：肿瘤抑制因子 BRCA2 是同源重组 (HR) 依赖性 DNA 修复的关键因素，失能突变会导致基因组不稳定和癌症易感性增加。BRCA2 的基本功能是促进 RAD51- 的成核。 ssDNA 丝是形成该中心 HR 中间体的限速步骤，参与与多种蛋白质（包括翻译后修饰剂和其他蛋白质）的相互作用。该项目的长期目标是阐明 BRCA2 及其结合伙伴在 HR 中的机制。具体目标是：具体目标 1：全长人类 BRCA2 蛋白的结构和功能是关键的 BRCA2 相互作用伙伴，DSS1 突变表型是 BRCA2 缺陷。 DSS1 在 BRCA2 介导的 HR 中发挥作用的精确机制仍有待确定，我们的初步数据表明 DSS1 对 BRCA2 结构的影响，因此 DSS1 生物化学被纳入 Subaim 1A，以将功能研究整合到结构方法中。 DSS1 使单链 DNA 上的单体 BRCA2 稳定以使 RAD51 丝形成核的假设获得全长人类 BRCA2 晶体结构的可能性在目前是不可行的。由于蛋白质的大小和片段性质，我们将使用 TEM 和冷冻电镜来确定 BRCA2 单独的整体结构以及与 DNA、DSS1 和 RAD51 的功能复合物的整体结构，以阐明其底物和主要成分的影响。 BRCA2 整体结构上的结合伙伴初步数据表明，我们可以可视化全长人类 BRCA2，其身份通过免疫金抗体染色和结合后的 C 端 His 标签得到确认。 DSS1 和 ssDNA，BRCA2 转变为开放的 C 形单体，与溶液中发现的单体或二聚体相比，具有显着的构象变化。利用我们的合作者 Al-Bassam 博士的非凡 EM 资源，我们将使用重建人类 BRCA2 的 3D 结构。 TEM 成像 (Subaim 1B) 和冷冻电镜 (Subaim 1C) 我们充分意识到图像重建伪影，并通过两个独立的重建工作来防止这种情况。阿尔·巴萨姆和斯塔尔伯格博士。