Project Summary Double-stranded DNA breaks (DSBs) represent a critical genotoxic lesion that is induced directly or indirectly by many anti-tumor agents including ionizing radiation, interstrand crosslinking agents, topoisomerase-targeted drugs, and agents that lead to replication forks stalling. Homologous recombination (HR) is a central pathway of genome maintenance that repairs DSBs caused by these agents. Defects in HR have dual significance for cancer. They lead to genomic instability and predispose to cancer. On the other hand, HR defects cause specific cellular vulnerabilities that can be exploited therapeutically. The overarching goal is to elucidate the mechanisms of HR. In this award period, we focus on the central HR intermediate, the displacement loop (D- loop), which is either targeted for dissociation by anti-recombination mechanisms or which is extended by DNA synthesis to transition further in the HR pathway. The Specific Aims are: (1) Mechanism and significance of D-loop editing by Topoisomerase 3a. Our published and unpublished work in S. cerevisiae showed that Top3-Rmi1 act as an anti-recombinase by targeting the D-loop intermediate. Using the tools and concepts developed in yeast, we will determine in Subaim 1A the biochemical mechanism involved using the yeast and human enzymes. In Subaim 1B, we will establish the significance of D-loop editing by TOPOIIIa in human cells. (2) Determine the pathways of D-loop editing. Using a newly developed general assay based on the proximity ligation principle to physically detect D-loops, we will conduct pathway analysis to determine which enzymes and pathways act on D-loops. In Subaim 2A, we will complete the pathway analysis in the budding yeast. Most of the effort is dedicated to Subaim 2B to determine the human enzymes and pathways that regulate D-loops levels. (3) Determine D-loop length and the factors controlling it. We have developed a novel assay to measure D-loop length at the single-molecule level. In Subaim 3A, we will validate the assay using reconstituted in vitro reactions with yeast and human recombination proteins. In Subaim 3B, we will define the mechanism, by which the motor protein Rdh54/Tid1 and its human homologs control D-loop length. In Subaim 3C, we will adapt this assay to human cells and determine D-loop length in human cells and test the human Rdh54/Tid1 paralogs for their effect on D-loop levels and length.