Mendiboure, N. | bioRxiv Science

bioRxiv2026

Rad55-Rad57 and Srs2 regulate homology search onset, coordination, reach and inactivation

DNA double-strand break (DSB) repair by homologous recombination entails the coordinated search for a homologous dsDNA molecule by two heterotypic Rad51-ssDNA filaments in eukaryotes. How homology search is regulated in cells remains largely unknown. Using genomic and molecular assays to track spatial chromatin organization and early recombination intermediates, we investigated the roles in homology search of two antagonistic regulators of Rad51-ssDNA filaments metabolism in S. cerevisiae: the Rad51 paralogs Rad55-Rad57 and the 3'-5' ssDNA translocase Srs2. Srs2 promoted the coordinated search between filaments on each DSB ends and inactivated homology search following homology identification. Rad55-Rad57 both stimulated the formation of Rad51-ssDNA filaments and protected them against disruption by Srs2. Together, Rad55-Rad57 and Srs2 enacted a structural proof-reading that resulted in stiffer Rad51-ssDNA filaments competent for genome-wide homology search. This work reveals multiple ways by which the control of Rad51-ssDNA filament metastability by the individual and joint activities of Rad55-Rad57 and Srs2 regulate homology search onset, coordination, reach and inactivation.

molecular biology↗

bioRxiv2023

Mechanism of homology search expansion during recombinational DNA break repair

Homology search catalyzed by a RecA/Rad51 nucleoprotein filament (NPF) is a central step of DNA double-strand break (DSB) repair by homologous recombination. How it operates in cells remains elusive. Here we developed a Hi-C-based methodology to map single-stranded DNA (ssDNA) contacts genome-wide in S. cerevisiae, which revealed two main homology search phases. Initial search conducted by short NPFs is confined in cis by cohesin-mediated chromatin loop folding. Progressive growth of stiff NPFs enables exploration of distant genomic sites. Long-range resection by Exo1 drives this transition from local to genome-wide search by providing ssDNA substrates for assembly of extensive NPFs. DSB end-tethering promotes coordinated homology search by NPFs formed on the two DSB ends. Finally, an autonomous genetic element on chromosome III engages the NPF and stimulates homology search in its vicinity. This work reveals the mechanism of the progressive and uneven expansion of homology search orchestrated by chromatin organizers, long-range resection, end-tethering, specialized genetic elements, and that exploits the stiff NPF structure conferred by Rad51 oligomerization. Highlights- Cohesin-mediated chromatin loops constrain homology search in cis for NPF regions close to the resection front - Stiffening of ssDNA by Rad51 enables genome-wide homology search by DSB-proximal sites - Exo1-mediated long-range resection promotes genome-wide homology search - DSB end-tethering promotes coordinated homology search by NPFs formed on both DSB ends - The recombination enhancer focuses homology search in its vicinity

genetics↗