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 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.