Assembly and Dynamics of Molecular Machines in Genome Maintenance

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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.