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(申请人提供)：肿瘤抑制基因BRCA2是依赖同源重组(HR)的DNA修复的关键因素，致残突变会增加基因组的不稳定性和癌症的易感性。BRCA2的基本功能是促进RAD51-ssDNA细丝的成核，这是形成这种中央HR中间体的限速步骤。BRCA2参与多种蛋白质的相互作用，包括翻译后修饰因子和其他调节因子。长期目标是阐明BRCA2及其结合伙伴在HR中的作用机制。本项目专注于一个特定的目标，即全长人BRCA2的整体结构和体系结构以及底物和结合伙伴对其调控。此特异性目的为：特异性目的1：人BRCA2全长蛋白的结构与功能。DSS1是BRCA2的关键交互伙伴。DSS1缺失和DSS1突变表现为BRCA2缺陷。然而，DSS1在BRCA2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。我们将检验DSS1稳定单链DNA上的单体BRCA2以形成RAD51细丝的假设。由于蛋白质的大小和节段性，目前获得全长人BRCA2晶体结构的可能性是不可行的。我们将使用透射电子显微镜和冷冻-EM来确定BRCA2单独的总体结构以及与DNA、DSS1和RAD51的功能络合物中的整体结构，以阐明其底物和主要结合伙伴对BRCA2整体结构的影响。初步数据显示，我们可以可视化全长的人BRCA2，其身份被免疫金抗体染色和靶向C末端His标签所证实。当与DSS1和ssDNA结合时，BRCA2转变为开放的C形单体，与溶液中的单体或二聚体相比有显著的构象变化。利用我们的合作者Al-Bassam博士非凡的EM资源，我们将使用透射电子显微镜成像(Subaim 1B)和冷冻EM(Subaim 1C)重建人类BRCA2的3D结构。我们充分意识到图像重建的伪影，我们通过Al-Bassam博士和Stahlberg博士的两个独立重建努力来防止这一点。