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: 10798482
• 负责人: Maria Spies
• 金额: $ 7.96万
• 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/10798482
• 关键词: 3-Dimensional; A-Form DNA; Acceleration; Affinity; Aging; Architecture; Award; Binding; Binding Proteins; Binding Sites; Biochemical; Biophysics; Blood; Brain Diseases; CAG repeat; Cancerous; Cell physiology; Cells; Collaborations; Complement; Complex; Cryoelectron Microscopy; DNA; DNA Binding Domain; DNA Damage; DNA Repair; DNA Replication Damage; DNA Structure; DNA biosynthesis; DNA lesion; DNA replication fork; DNA-Directed DNA Polymerase; Data; Data Analyses; Decision Making; Defect; Event; Fluorescence Microscopy; Funding; G-Quartets; Genome; Genome Stability; Goals; Grant; Hand; Human; Huntington Disease; Kinetics; Maintenance; Malignant Neoplasms; Manuscripts; Modeling; Molecular; Molecular Conformation; Molecular Machines; Neck; Nerve Degeneration; Neurodegenerative Disorders; Normal Cell; Nucleoproteins; Paper; Parents; Pathogenesis; Pathway interactions; Photometry; Poly T; Positioning Attribute; Primates; Process; Proteins; Radiation induced damage; Regulation; Request for Applications; Resected; Resistance development; Signal Transduction; Single-Stranded DNA; Structure; Syndrome; Total Internal Reflection Fluorescent; Traction; United States National Institutes of Health; Universities; Variant; Visualization; Work; biophysical analysis; c-myc Genes; chemotherapy; computerized data processing; crosslink; data structure; follow-up; gain of function; gain of function mutation; helicase; homologous recombination; laser tweezer; melting; molecular dynamics; molecular recognition; mutant; new therapeutic target; novel; optic tweezer; overexpression; particle; programs; reconstitution; reconstruction; repaired; replication factor A; single molecule; telomere

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), 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 the two projects is a dynamic complex between ssDNA binding protein RPA (Replication Protein A) and DNA, including ssDNA at resected DNA breaks, damaged replication forks, DNA repair intermediates, structures arising at DNA repeats, and G-quadruplexes. Our single-molecule and biochemical data suggest that the architecture and the dynamics of the RPA-DNA complexes is regulated by specific RPA partners and in differences in RPA engagement to different DNAs. The structures and architectures of these complexes remain elusive. Recent advances in CryoEM allowing us to advance a structural understanding of the RPA-DNA complexation on unstructured and telomeric DNA. We are also pursuing structures of RPA-telomere-hnRNPA1 complex, and FANCJ helicase bound to telomeric and cMyc G-quadruplexes. While we achieved a significant experimental traction, data processing remains a bottle neck for our CryoEM work. This application requests funds for acquisition of the Exxact workstation configured specifically for GPU accelerated CryoEM single particle 3D reconstruction, which will allow us to consolidate in house data processing and structure determination. Progress on the structures containing RPA and FANCJ complexes will help us to build a completely new picture of the nexus between RPA configuraiotnal dynamics and shuttling of the RPA-containing complexes into specific genome maintenance pathways.

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。项目2调查 多用途DNA修复解旋酶及其通过难以复制协调DNA复制的能力 区域，因此也有助于基因组的稳定。 这两个项目都利用了单分子全内反射荧光显微镜(SmTIRFM)， 光学镊子和荧光显微镜(CTFM)、质量光度测定和生化重建 可视化和量化协调HR的核蛋白复合体的动态组装和重塑 以及处理替代的DNA结构。在这两种情况下，我们研究的所有过程中的关键中间体 项目是单链DNA结合蛋白RPA(复制蛋白A)和DNA之间的动态复合体，包括 切除的DNA断裂处的单链DNA，受损的复制叉处，DNA修复中间产物，在DNA处产生的结构 重复和G-四联体。我们的单分子和生化数据表明， RPA-DNA复合体的动力学受特定的RPA伙伴和RPA中的差异调节 与不同的DNA接触。这些建筑群的结构和建筑仍然难以捉摸。 低温EM的最新进展使我们对RPA-DNA络合的结构有了更深入的了解 在非结构和端粒DNA上。我们还在寻找RPA-端粒-hnRNPA1复合体的结构，以及 FANCJ解旋酶与端粒和cMyc G-四链结合。虽然我们完成了一项重要的实验 牵引，数据处理仍然是我们低温电子显微镜工作的瓶颈。此应用程序请求资金用于 获得专为GPU加速的CryoEM单粒子3D配置的Exxact工作站 重建，这将使我们能够巩固内部数据处理和结构确定。进展 关于含有RPA和FANCJ络合物的结构将有助于我们构建一幅全新的图景 RPA构型动力学和含RPA的复合体穿梭到特定的 基因组维持途径。