Double strand DNA breaks in the cell are repaired primarily by the Homologous Recombination (HR) system. Defective or unregulated HR results in chromosome rearrangements leading to genomic instability, which in turn results in a variety of cancers and hereditary disorders. Formation of the Rad51 filament on single- stranded DNA (ssDNA) is one of the key events in the HR pathway and is under tight regulation by both pro- and anti-recombinogenic mediator proteins. Our long-term goal is to precisely determine the dynamics of Rad51 nucleoprotein filament formation and its disassembly on ssDNA and the mechanism by which various pro- and anti-HR mediator proteins alter these dynamics to influence HR. Technical limitations have hindered the study of Rad51 nucleoprotein filament formation on single-stranded DNA (ssDNA). We propose to overcome these obstacles through the development of fluorescent Rad51 and RPA probes which, when bound to ssDNA will undergo a change in fluorescence. We will be utilizing the unnatural amino acid incorporation strategy to develop these probes which will be combined with stopped flow kinetic assays to monitor the dynamics of the nucleoprotein filament in real-time. Since our probes are direct reporters of Rad51/RPA association/dissociation, we will utilize this fluorescent tool-kit to investigate how Rad51 and RPA bind to ssDNA when they are both present in a multi-protein experimental setup. Significance of studying Rad51 filament formation on both ssDNA and dsDNA substrates is highlighted by our recent findings that the bound-DNA context of a Rad51 filament diametrically controls the activity of Srs2. Srs2 is an anti-recombinase that clears Rad51 nucleoprotein filaments from ssDNA and as a helicase is capable of unwinding dsDNA. Physical interaction between Rad51 and Srs2 is required to dismantle the nucleoprotein filament and is observed strictly on ssDNA. When Rad51 is bound on dsDNA, it inhibits the DNA unwinding activity of Srs2 through the very same physical interaction interface. The Specific Aims are to: (1) Develop a real-time fluorescence-based tool kit to measure nucleoprotein filament dynamics on ssDNA. (2) Determine the mechanism behind the DNA-context dependent regulation of Srs2 activity by Rad51. We will obtain kinetic parameters for the various steps in the nucleoprotein assembly and disassembly process and uncover the molecular details of the interaction between Srs2 and Rad51. These studies will enable us to build a precise mechanistic model of how Rad51 nucleoprotein filaments are formed and how mediator proteins alter its dynamics. This information is critical in understanding the role of mediator proteins in HR associated defects, and to uncover why mutations in these proteins lead to genomic instability, cancers and associated genetic disorders. In addition to addressing these research questions, we will develop multiple research projects well suited to engage undergraduate students in bio-medically relevant research at Utah State University.