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2调查 多用途DNA修复解旋酶和它们通过难以复制的DNA复制来协调DNA复制的能力 这也有助于基因组的稳定性。 这两个项目都利用单分子全内反射荧光显微镜（smTIRFM）， 光镊和荧光显微镜（CTFM），质量光度法和生化重建， 可视化和量化协调HR的核蛋白复合物的动态组装和重塑 和处理不同的DNA结构。在我们研究的所有过程中的关键中间体， 项目是ssDNA结合蛋白RPA（复制蛋白A）和DNA之间的动态复合物，包括 ssDNA在切除的DNA断裂，损坏的复制叉，DNA修复中间体，结构出现在DNA 重复和G-四链体。我们的单分子和生化数据表明， RPA-DNA复合物的动力学受特定RPA伴侣的调节， 不同的DNA。这些复合物的结构和架构仍然难以捉摸。 CryoEM的最新进展使我们能够推进RPA-DNA复合的结构理解 非结构化和端粒DNA上。我们还在研究RPA-端粒-hnRNPA 1复合物的结构， FANCJ解旋酶与端粒和cMyc G-四链体结合。虽然我们取得了重大的实验成果， 牵引力，数据处理仍然是我们的CryoEM工作的瓶颈。该申请要求资金用于 获取专门为GPU加速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 对含有RPA和FANCJ复合物的结构的研究将有助于我们建立一个全新的图像， RPA构型动力学与含RPA的复合物穿梭到特定的 基因组维持途径。

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.