Project Summary Homologous recombination (HR) maintains genomic stability through high-fidelity repair of DNA double-stranded breaks (DSB) and other complex DNA damage that is induced directly or indirectly by common anti-tumor agents including ionizing radiation, topoisomerase-targeted drugs, interstrand crosslinking agents, and those causing replication forks stalling. HR defects bear dual significance for cancer by first leading to genomic instability and increased cancer predisposition. Moreover, HR defects cause specific cellular vulnerabilities that can be exploited therapeutically either by traditional DNA damage-based treatment or by targeted treatment for example by poly(ADP-ribose) polymerase inhibition. The overarching goal is to elucidate the mechanisms of HR. This application focuses on a central HR intermediate, the displacement loop (D-loop), which represents the branchpoint for the HR sub-pathways. The Specific Aims are: Aim 1: Define D-loop length in cells and the role of Rdh54 in D-loop metabolism We will take biochemical (Aim 1A) and genetic approaches (Aim 1B) to determine for the first time the length of D-loops using a newly developed assay that maps D-loops at single molecule resolution. In Aim 1C, we will determine the role of Rdh54 in controlling D-loop length and crossover outcome. Aim 2: Delineate the role of human RAD54B in HR In testing the model of functional cooperation with RAD54, we will determine the role of RAD54B in HR by using a biochemical approach to determine its role in RAD51-mediated recombination using established and newly developed assays (Aim 2A). In Aim 2B, we extend these studies to human cells based on preliminary data showing a specific role of RAD54B in synthesis-dependent strand annealing (SDSA). In Aim 2C, we will adapt novel assays to human cells, determine D-loop length, and test the effect of RAD54B on D-loop levels and length to determine the mechanisms of crossover avoidance. Aim 3: Determine the roles of RECQ1 and RECQ5 in D-loop editing and crossover control D-loops are a central HR intermediate and highly dynamic. We surmise that D-loops encompass a diverse set of structures explaining the existence of multiple D-loop editing pathways. Based on exciting preliminary data, we will focus on RECQ1 as a novel player acting in concert with RECQ5 in SDSA. We will determine the mechanisms RECQ1 and RECQ5 in SDSA using a biochemical in vitro reconstitution approach (Aim 3A) complemented by genetic and cell biological approaches in Aim 3B.