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.

抽象的 为了维持稳定的基因组，细胞执行了准确，及时的复制程序并修复此类 有害的DNA病变是双链断裂，链间交叉链接和损坏的复制叉。 父母NIH R35GM131704 MIRA GRANT（PI：SPIES）的项目1研究了分子机械 同源重组（HR），这是一种提供最准确修复这些方法的蜂窝过程 有害的DNA病变和损坏的复制叉，从而有助于正常的基因组稳定性 细胞，但也有助于癌细胞发展对放射线和DNA损害化疗的抗性。我们 正在建立对人力资源及其法规中核心步骤的定量描述，这将利用 蛋白质可塑性和分子识别中构象动力学的重要性。项目2调查 多功能DNA修复解酶及其通过难以复制来协调DNA复制的能力 地区，因此也有助于基因组稳定性。这两个项目都使用单分子总内部反思 荧光显微镜（SMTIRFM）可视化和量化动态组装和重塑 核蛋白复合物可协调HR和替代DNA结构的处理。质量光度法是 强大的新技术将通过允许我们量化来为我们的测量增加一个新的维度 构成核蛋白复合物的分子物种的分布。 该申请要求资金以获取Refeyn Onemp质量光度计，该乐器是 使用干涉散射（ISCAT，又名ISCAMS），用于无标记的个体分子质量检测 溶液中的大分子。该系统将促进我们执行在 家长R35奖，并将大大增强我们方法的严格性。质量光度法将高度 与SMTIRFM分析的互补性，因为这将使我们能够直接评估寡聚状态的分布 RAD51重组酶及其突变体的突变体在低生理上以降低Rad51-Rad51界面改变 我们在SMTIRFM研究中使用的相关浓度。我们将能够定量评估 RAD51的低聚物状态以及含RAD51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RAD51-RAD51结合亲和力的小分子抑制剂的存在）。我们将能够 明确确定含有的配合物的平衡分离常数和组成 RAD51和肿瘤抑制剂BRCA2的Rad51相互作用片段，并将跟随形成 rad51核蛋白丝的方式是与我们的SMTIRFM测量值互补的。添加 单分子ISCAT测量将有助于我们建立全新的New图片 RAD51单体 - 理学界面和人力资源中的细丝特性的扰动 复制。