Homologous recombination faithfully restores the sequence information interrupted by a DNA double-strand break by referencing an intact DNA molecule as a template for repair DNA synthesis. DNA synthesis is primed from 3'-OH end of the invading DNA strand in the displacement loop (D-loop). Here, we describe a simple and quantitative proximity ligation-based assay to study the initiation of homologous recombination-associated DNA synthesis initiated at the D-loop and final product formation. The D-loop extension assay overcomes the semiquantitative nature and some limitations of the current PCR-based technique and facilitates the study of the recombination-associated DNA synthesis.

Displacement loops (D-loops) are pivotal intermediates of homologous recombination (HR), a universal DNA double strand break (DSB) repair pathway. We developed a versatile assay for the physical detection of D-loops in vivo, which enabled studying the kinetics of their formation and defining the activities controlling their metabolism. Nascent D-loops are detected within 2 h of DSB formation and extended in a delayed fashion in a genetic system designed to preclude downstream repair steps. The majority of nascent D-loops are disrupted by two pathways: one supported by the Srs2 helicase and the other by the Mph1 helicase and the Sgs1-Top3-Rmi1 helicase-topoisomerase complex. Both pathways operate without significant overlap and are delineated by the Rad54 paralog Rdh54 in an ATPase-independent fashion. This study uncovers a layer of quality control of HR relying on nascent D-loop dynamics.