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

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

抽象的为了维持稳定的基因组，细胞执行准确且及时的复制程序并修复这些基因组。有害的 DNA 损伤，如双链断裂、链间交联和复制叉受损。 NIH R35GM131704 MIRA 资助的项目 1（PI：Spies）研究了分子机制同源重组（HR），一种细胞过程，提供最准确的方法来修复这些有害的 DNA 损伤和复制叉受损，从而有助于正常情况下的基因组稳定性细胞，还可以帮助癌细胞产生对辐射和 DNA 损伤化疗的抵抗力。我们正在建立人力资源核心步骤及其监管的定量描述，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。 MIRA 奖项目 1 采用单分子全内反射荧光显微镜 (smTIRFM)、相关光镊和荧光显微镜 (CTFM)、质量光度测定和生化重构以可视化和量化核蛋白的动态组装和重塑协调 HR 和替代 DNA 结构加工的复合物。所有流程的关键中间体我们在这个项目下研究的是ssDNA结合蛋白RPA（复制蛋白A）之间的动态复合物， RAD51 重组酶和 DNA。在 HR 中，为了保护停滞和受损的 DNA 复制叉，RPA 和 RAD51 竞争 ssDNA 结合，并且这种竞争受到严格监管。作为一名暑期学生，Sabryn Labenz 女士（北爱荷华大学）将利用质量光度测定法来收集野生型 RAD51 重组酶以及 RAD51 突变体的寡聚状态分布数据改变了原体-原体相互作用界面。 RAD51 形成不同尺寸低聚物的能力将与 RAD51 核蛋白丝形成速率（由 CTMF 测定）和 RAD51 取代 ssDNA 结合蛋白 RPA 并进行 DNA 链交换反应的能力。 RAD51 寡聚状态的计数和核蛋白形成的定量将使 Sabryn 改进 RAD51/RPA 竞赛的理论模型。