Structure and Function of the Tumor Suppressor Protein BRCA2

肿瘤抑制蛋白 BRCA2 的结构和功能

• 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
• 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

 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（由应用提供）：肿瘤抑制器BRCA2是同源重组（HR）依赖性DNA修复和残疾突变的关键因素，导致基因组不稳定性增加和癌症倾向。 BRCA2的基本功能是促进Rad51-SsDNA细丝的成核，这是该中央HR中间体形成的速率限制步骤。 BRCA2与多种蛋白质进行相互作用，包括翻译后修饰剂和其他调节剂。长期目标是阐明BRCA2及其在人力资源中的结合伙伴的机制。该项目侧重于一个特定目标，全长人BRCA2的整体结构和结构及其对底物和约束伙伴的调制。该特定目的是：特定目标1：全长人BRCA2蛋白的结构和功能。 DSS1是关键的BRCA2相互作用伙伴。 DSS1耗竭和DSS1突变表观A BRCA2缺陷。但是，DSS1在BRCA2介导的HR中起作用的精度机制仍有待确定。我们的初步数据表明DSS1对BRCA2架构的影响，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。我们将测试DSS1在ssDNA上稳定单体BRCA2的假设，以形成rad51丝形成。由于蛋白质的大小和分段性质，目前无法获得全长人BRCA2的晶体结构的可能性。我们将使用TEM和冷冻EM来确定单独的BRCA2和与DNA，DSS1和RAD51的功能复合物的总体结构，以阐明其底物和主要结合伙伴对BRCA2整体结构的影响。初步数据表明，我们可以可视化全长的人BRCA2，其身份通过免疫原抗体染色确认并瞄准其标签的C末端。结合DSS1和ssDNA后，BRCA2转变为开放的C形单体，其与溶液中的单体或二聚体发生了显着的会议变化。使用我们的合作者Al-Bassam博士的非凡资源，我们将使用TEM成像（Subaim 1B）和Cryo-Em（Subaim 1C）重建人BRCA2的3D结构。我们完全意识到图像重建工件，我们通过DRS进行了两项独立的重建工作来保护这一问题。 Al-Bassam和Stahlberg。