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: (1) Define D-loop length in cells and the role of Rdh54 in D-loop metabolism. (2) Delineate the role of human RAD54B in HR. (3) Determine the roles of RECQ1 and RECQ5 in D-loop editing and crossover control. All aims require the use of the requested refrigerated incubator/shaker used for protein production by insect cells in this application.