ABSTRACT Efficient genome maintenance is a double-edged sword. Accurate repair of DNA lesions and damaged replication forks promotes genome stability, which is a key to avoiding cancer, aging and neurodegenerative diseases associated with DNA repeat expansion. My research program under the parent NIH R35GM131704 MIRA grant (PI: Spies) investigates the molecular and structural mechanisms by which the intermediates of DNA metabolism bound by Replication Protein A (RPA) are channeled into DNA repair, protection of DNA replication forks, homologous recombination, DNA damage tolerance and signaling. Our goal is to reconstitute (in vitro and in singulo) and manipulate (in vivo) the elaborate network of DNA repair mechanisms, and to understand how cells “choose” between accurate DNA repair and “rogue” mechanisms that destabilize the genome. Ability to visualize and investigate biologically important molecules individually, in real time, and under physiological conditions is critical for developing mechanistic understanding of how cells work and communicate, how molecular machines assemble and function, and, in general, how physiological functions emerge from the chaos of stochastic molecular events and interactions. All projects under the parent MIRA award utilize single- molecule total internal reflection fluorescence microscopy (smTIRFM), correlated optical tweezers and fluorescence microscopy (CTFM), mass photometry, structural biology (CryoEM) and biochemical reconstitutions to visualize and quantify the dynamic assembly and remodeling of the nucleoprotein complexes coordinating DNA repair events, homologous recombination and processing of non-canonical DNA structures. While my lab has been successful in broadening the scope of state-of-the-art single-molecule approaches to study macro-molecular interactions, their dynamics and inhibition using smTIRFM, surface-tethering required for this approach makes it impossible to rapidly screen experimental conditions, build fluorescence and Forster resonance energy transfer (smFRET) distributions. This application requests funds for acquisition of the EI-FLEX, a single-molecule fluorescence spectrometer which has a capacity to simultaneously and rapidly record (i) fluorescence and FRET from single molecules in solution, and (ii) fluorescence decay correlations which inform on complex sizes. Such information on the complexes containing RPA, alt-RPA, FANCJ, REV1 and PARP1 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.