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: 10808780
• 负责人: Maria Spies
• 金额: $ 1.47万
• 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/10808780
• 关键词: ATP Hydrolysis; Affect; Award; Binding; Binding Proteins; Biochemical; Cancerous; Cell physiology; Cells; Complex; DNA; DNA Damage; DNA Replication Damage; DNA Structure; DNA biosynthesis; DNA lesion; DNA replication fork; Data; Enzymes; Fanconi' s Anemia; Filament; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Genetic Recombination; Genome; Genome Stability; Grant; Human; Iowa; Maintenance; Malignant Neoplasms; Mediating; Mediator; Modeling; Molecular; Molecular Conformation; Molecular Machines; Normal Cell; Nucleoproteins; Parents; Participant; Pharmaceutical Preparations; Photometry; Play; Process; Proliferating; Protein Dynamics; Proteins; Protomer; Rad51 recombinase; Radiation induced damage; Reaction; Regulation; Research; Resistance development; Single-Stranded DNA; Testing; Theoretical model; Therapeutic; United States National Institutes of Health; Universities; Visualization; Work; cancer therapy; carcinogenesis; chemotherapeutic agent; chemotherapy; crosslink; helicase; homologous recombination; improved; laser tweezer; molecular dynamics; molecular recognition; mutant; optic tweezer; permissiveness; prevent; programs; recombinase; reconstitution; repaired; replication factor A; single molecule; summer student

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 1 under this MIRA award utilizes single-molecule total internal reflection fluorescence microscopy (smTIRFM), correlated optical tweezers and fluorescence microscopy (CTFM), mass photometry and biochemical reconstitutions to visualize and quantify the dynamic assembly and remodeling of the nucleoprotein complexes coordinating HR and processing of alternative DNA structures. The key intermediate in all processes we study under this project is a dynamic complex between ssDNA binding protein RPA (Replication Protein A), RAD51 recombinase and DNA. In HR and in protection of stalled and damaged DNA replication forks, RPA and RAD51 compete for the ssDNA binding and this competition is tightly regulated. As a summer student, Ms. Sabryn Labenz (University of Northern Iowa) will utilize mass photometry to collect data on the oligomeric state distribution of the wild type RAD51 recombinase, as well as RAD51 mutants that have altered protomer-protomer interaction interface. The ability of RAD51 to form oligomers of different sizes will be correlated with the rate of the RAD51 nucleoprotein filament formation (determined by CTMF) and the ability of RAD51 to displace the ssDNA binding protein RPA and carry out the DNA strand exchange reactions. The enumeration of the RAD51 oligomeric states and quantification of the nucleoprotein formation will enable Sabryn to improve the theoretical model for the RAD51/RPA competition.

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样的 有害的DNA损伤，如双链断裂、链间交联和复制叉子损坏。 母公司NIH R35GM131704 Mira Grant(PI：SPIES)的项目1研究了 同源重组(HR)，一个细胞过程，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，从而有助于正常人类基因组的稳定 但也有助于癌细胞对辐射和破坏DNA的化疗产生抵抗力。我们 正在对人力资源及其监管的核心步骤进行量化描述，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。 该Mira奖下的项目1利用单分子全内反射荧光显微镜 (SmTIRFM)，相关光学镊子和荧光显微镜(CTFM)，质量光度学和 用于可视化和量化核蛋白动态组装和重塑的生化重组 协调HR和处理替代DNA结构的复合体。所有流程中的关键中间体 我们在这个项目下研究的是一个动态的SSDNA结合蛋白RPA(复制蛋白A)， RAD51重组酶和DNA。在HR中以及在保护停滞和损坏的DNA复制叉时，RPA和 RAD51竞争单链DNA结合，这种竞争受到严格的监管。 作为暑期学生，Sabryn Labenz女士(北爱荷华州大学)将利用质量光度学来收集 野生型RAD51重组酶的寡聚态分布数据，以及RAD51突变体 改变了原-原相互作用界面。RAD51形成不同大小低聚物的能力 将与RAD51核蛋白细丝形成速度(由CTMF确定)和 RAD51能够取代单链DNA结合蛋白RPA并进行DNA链交换反应。 RAD51寡聚态的计数和核蛋白形成的量化将使 Sabryn改进了RAD51/RPA竞争的理论模型。