Project Summary Our chromosomes are continually bombarded with a variety of insults, resulting in damage that must be repaired. By necessity, cells have evolved mechanisms to detect and repair broken strands of DNA, thereby preventing loss of important genetic information. Double-stranded DNA breaks (DSBs) are a type of damage that can result in particularly disastrous outcomes. If not corrected, DSBs can lead to gross chromosomal rearrangements, which are the hallmark of all forms of cancer. Indeed, defects in HR-related proteins are associated with several severe genetic diseases. Patients with these diseases often exhibit a strong predisposition for developing cancers due to a loss of genome integrity. Surprisingly, DNA replication is the primary source of DSBs, and as a consequence rapidly growing cells are especially dependent upon homologous DNA recombination for survival. This dependence upon homologous recombination for the survival of rapidly growing cells highlights the potential for using recombination inhibitors as highly selective anti-cancer therapies. To exploit the clinical potential of homologous recombination inhibitors it will be essential that we more fully understand the molecular underpinnings of the proteins that are involved in regulating and controlling recombination. To help better understand the molecular basis of homologous DNA recombination we have developed powerful new experimental platforms that allow us to directly visualize hundreds of individual DNA molecules at the single molecule level. We are utilizing these unique research tools to probe the fundamental basis for protein-nucleic acid interactions, with emphasis placed upon understanding reactions relevant to human biology and disease. We have used these assays to study human RAD51, which binds to single- stranded DNA forming a key recombination intermediate called the presynaptic complex. Here, we will assess how complexes containing the tumor suppressor protein complexes BRCA1-BARD1, BRCA2- DSS1, and PALB2 promote homologous recombination by regulating the activities of the RAD51 presynaptic complex. We will also examine how the protein RAD52, which newly recognized as an important target for anti-cancer drugs, interacts with RAD51, BRCA1-BARD1, BRCA2-DSS1, and PALB2. We will also study the protein RADX, which is emerging as a key player in genome integrity which functions to downregulate RAD51 activity. We will accomplish these goals by directly visualizing these processes in real-time using optical microscopy. These studies offer the potential for significant new insights into how BRCA1-BARD1, BRCA2-DSS1, PALB2 and RAD52 regulate homologous recombination and support human genome integrity.