Assembly and Dynamics of Molecular Machines in Genome Maintenance

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1在此MIRA奖利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）、质量光度法和 生物化学重建，以可视化和量化核蛋白的动态组装和重塑 协调HR和处理替代DNA结构的复合物。所有过程中的关键中间体 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， RAD 51重组酶和DNA。在HR和保护停滞和受损的DNA复制叉中，RPA和 RAD 51竞争ssDNA结合，并且这种竞争受到严格调控。 作为一名暑期学生，Sabryn Labenz女士（北方爱荷华州大学）将利用质量测光收集 关于野生型RAD 51重组酶以及 已经改变了原体-原体相互作用界面。RAD 51形成不同大小的寡聚物的能力 将与RAD 51核蛋白丝形成的速率（由CTMF测定）和 RAD 51置换ssDNA结合蛋白RPA并进行DNA链交换反应的能力。 RAD 51寡聚体状态的计数和核蛋白形成的定量将使得 Sabryn改进了RAD 51/RPA竞赛的理论模型。

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.