Mechanism of homology search expansion during recombinational DNA break repair

Abstract

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 inS. cerevisiae, which revealed two main homology search phases. Initial search conducted by short NPFs is confined incisby 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 incisfor 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

All who wander are not lost: the search for homology during homologous recombination

“…This group of proteins includes the Condensin, COHESIN, SMC5/6 proteins, and RecN in prokaryotes [ 99 ]. Recently, it has been shown that COHESIN acts as a topological barrier to promote local homology search and inhibit long-range homology searches that can lead to genomic rearrangements [ 90 , 100 ]. While the structural contribution of these proteins has been identified, more work will be needed to understand how their role in the topological insulation of specific domains may contribute to the rates of homologous DNA selection.…”

The translocation activity of Rad54 reduces crossover outcomes during homologous recombination

“…Formation of the D-loop during HR is a dynamic process that requires the selection of an appropriate template and stabilization of strand pairing events in a process called the homology search ( 7–11 , 16–22 ). A successful homology search leads to the extension of D-loops, which allows the transition to the DNA repair and resolution phase of HR ( 23 ).…”