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（由申请人提供）：肿瘤抑制因子BRCA 2是同源重组（HR）依赖性DNA修复的关键因子，失能突变导致基因组不稳定性增加和癌症易感性增加。BRCA 2的基本功能是促进RAD 51-ssDNA丝的成核，这是形成该中心HR中间体的限速步骤。BRCA 2参与与多种蛋白质的相互作用，包括翻译后修饰物和其他调节剂。长期目标是阐明BRCA 2及其结合伴侣在HR中的机制。该项目侧重于一个特定目标，全长人类BRCA 2的整体结构和架构及其底物和结合伴侣的调节。具体目标1：全长人BRCA 2蛋白的结构和功能。DSS 1是BRCA 2相互作用的重要伙伴。DSS 1缺失和DSS 1突变表型复制BRCA 2缺陷。然而，DSS 1在BRCA 2介导的HR中发挥作用的确切机制仍有待确定。我们的初步数据表明DSS 1对BRCA 2结构的影响，因此将DSS 1生物化学作为Subaim 1A纳入，以将功能研究整合到结构方法中。我们将检验DSS 1稳定ssDNA上的单体BRCA 2以使RAD 51丝形成成核的假设。由于蛋白质的大小和节段性质，目前获得全长人BRCA 2的晶体结构的可能性是不可行的。我们将使用TEM和cryo-EM来确定BRCA 2单独的整体结构以及与DNA，DSS 1和RAD 51的功能复合物，以阐明其底物和主要结合伴侣对BRCA 2整体结构的影响。初步数据显示，我们可以可视化全长人BRCA 2，其身份通过免疫金抗体染色和靶向C-末端His标签确认。在结合DSS 1和ssDNA后，BRCA 2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著的构象变化。利用我们的合作者Al-Bassam博士非凡的EM资源，我们将使用TEM成像（Subaim 1B）和cryo-EM（Subaim 1C）重建人类BRCA 2的3D结构。我们充分意识到图像重建伪影，我们通过Al-Bassam和斯塔尔贝格博士的两项独立重建工作来防止这种情况。