Assembly and Dynamics of Molecular Machines in Genome Maintenance

基因组维护中分子机器的组装和动力学

• 批准号: 10377656
• 负责人: Maria Spies
• 金额: $ 17.25万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10377656
• 关键词: ATP Hydrolysis; Affect; Affinity; Amino Acids; Award; BRCA2 gene; Binding; Biological Assay; C-terminal; Cancerous; Cell physiology; Cells; Complex; Cryoelectron Microscopy; Crystallography; DNA; DNA Damage; DNA Repair; DNA Replication Damage; DNA Structure; DNA biosynthesis; DNA lesion; DNA replication fork; DNA-Protein Interaction; Data; Detection; Dimensions; Dissociation; Equilibrium; Filament; Fluorescence Microscopy; Funding; Gap Junctions; Genetic Polymorphism; Genetic Recombination; Genome; Genome Stability; Grant; Heterogeneity; Human; Individual; Iowa; Label; Letters; Link; Macromolecular Complexes; Maintenance; Malignant Neoplasms; Measurement; Mediator of activation protein; Medicine; Microscopy; Modeling; Molecular; Molecular Conformation; Molecular Machines; Monitor; Normal Cell; Nucleic Acids; Nucleoproteins; Parents; Photometry; Physiological; Property; Proteins; Protomer; RAD52 gene; Rad51 recombinase; Radiation induced damage; Regulation; Reporting; Request for Applications; Research; Resistance development; Sampling; Signal Transduction; Support Contracts; Surface; System; Techniques; Testing; Time; Tumor Suppressor Proteins; United States National Institutes of Health; Universities; anti-cancer therapeutic; base; chemotherapy; college; crosslink; dimer; experimental study; falls; genome integrity; genotoxicity; helicase; homologous recombination; improved; inhibitor/antagonist; instrument; macromolecule; molecular dynamics; molecular mass; molecular recognition; monomer; mutant; novel; nuclease; preservation; prevent; programs; repaired; single molecule; small molecule; small molecule inhibitor; stoichiometry; tool

ABSTRACT To maintain stable genomes, cells carry out an accurate and timely replication program and repair such deleterious DNA lesions as double-stranded breaks, inter-strand crosslinks, and damaged replication forks. Project 1 of the parent NIH R35GM131704 MIRA grant (PI: Spies) investigates the molecular machinery of homologous recombination (HR), a cellular process that provides the most accurate means to repair of these deleterious DNA lesions and damaged replication forks, and thereby contributes to genome stability in normal cells, but also helps cancerous cells to develop resistance to radiation and DNA-damaging chemotherapy. We are building a quantitative description of the central step in HR and its regulation, which will draw on the importance of protein plasticity and conformational dynamics in molecular recognition. Project 2 investigates multipurpose DNA repair helicases and their ability to coordinate DNA replication through difficult to replicate regions, thus also contributing to genome stability. Both projects utilize single-molecule total internal reflection fluorescence microscopy (smTIRFM) to visualize and quantify the dynamic assembly and remodeling of the nucleoprotein complexes coordinating HR and processing of alternative DNA structures. Mass photometry is a powerful new technique that will add a new dimension to our measurements by allowing us to quantify the distributions of the molecular species constituting nucleoprotein complexes. This application requests funds for acquisition of the Refeyn OneMP Mass Photometer, an instrument that uses interferometric scattering (iSCAT, aka iScaMS) for label-free detection of molecular mass of individual macromolecules in solution. This system will facilitate our capacity to perform the experiments proposed in the parent R35 award and will significantly enhance the rigor of our approaches. Mass photometry will be highly complementary to smTIRFM analyses, as it will allow us to directly assess the distributions of oligomeric states of the RAD51 recombinase and its mutants with altered RAD51-RAD51 interface at low, physiologically relevant concentrations we use in the smTIRFM studies. We will be able to quantitatively evaluate how the oligomeric states of RAD51 and heterogeneity of the RAD51- and RPA-containing complexes change with changing the solution conditions (including conditions permitting and restricting ATP hydrolysis, and the presence of small molecule inhibitors that may affect the RAD51-RAD51 binding affinity). We will be able to unambiguously determine the equilibrium dissociation constants for and composition of complexes containing RAD51 and the RAD51-interacting fragments of the tumor suppressor BRCA2, and will follow the formation of the RAD51 nucleoprotein filament in a manner complementary to our smTIRFM measurements. Addition of the single-molecule iSCAT measurements will help us to build a completely new picture of the nexus between perturbations of the RAD51 monomer-monomer interface and the filament properties in HR and during replication.

摘要 为了维持稳定的基因组，细胞进行准确和及时的复制程序，并修复这种基因组。 有害的DNA损伤，如双链断裂、链间交联和受损的复制叉。 美国国立卫生研究院R35 GM 131704 MIRA资助的项目1（PI：Spies）研究了 同源重组（HR），这是一种细胞过程，提供了最准确的手段来修复这些 有害的DNA损伤和受损的复制叉，从而有助于正常细胞中的基因组稳定性。 细胞，而且还有助于癌细胞对辐射和DNA损伤化疗产生抵抗力。我们 正在建立一个定量描述的核心步骤，人力资源及其监管，这将借鉴 蛋白质可塑性和构象动力学在分子识别中的重要性。项目2调查 多用途DNA修复解旋酶和它们通过难以复制的DNA复制来协调DNA复制的能力 这也有助于基因组的稳定性。这两个项目都利用单分子全内反射 荧光显微镜（smTIRFM），以可视化和量化的动态组装和重塑的 核蛋白复合物协调HR和替代DNA结构的加工。质量测光是一种 强大的新技术，将增加一个新的层面，我们的测量，使我们能够量化的 构成核蛋白复合物的分子种类的分布。 该申请要求获得Refeyn OneMP质量光度计的资金，该仪器 使用干涉散射（iSCAT，aka iScaMS）对单个样品的分子量进行无标记检测 溶液中的大分子。这一系统将有助于我们进行本报告中提出的实验。 R35大奖，并将大大提高我们的方法的严谨性。质量测光将是高度 补充smTIRFM分析，因为它将使我们能够直接评估低聚状态的分布 具有改变的RAD 51-RAD 51界面的RAD 51重组酶及其突变体在低的、生理的 我们在smTIRFM研究中使用的相关浓度。我们将能够定量评估 RAD 51的寡聚状态和含有RAD 51和RPA的复合物的异质性随着 改变溶液条件（包括允许和限制ATP水解的条件，以及 可能影响RAD 51-RAD 51结合亲和力的小分子抑制剂的存在）。我们将能够 明确地确定平衡解离常数和复合物的组成， RAD 51和RAD 51相互作用的肿瘤抑制因子BRCA 2片段，并将遵循 RAD 51核蛋白丝的方式补充我们的smTIRFM测量。添加 单分子iSCAT测量将帮助我们建立一个全新的关系图 RAD 51单体-单体界面的扰动和HR中的长丝性质以及在 复制的