Mechanism of homology search expansion during recombinational DNA break repair

Abstract

AbstractHomology 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