Cohesin drives chromatin scanning during RAD51-mediated homology search

Cohesin folds mammalian genomes into chromatin loops, whose functional roles are under debate. We report that Cas9 breaks induce de novo formation of chromatin loops, with the break positioned at the loop base. These break-anchored loops form only in the S/G2 phases and occur preferentially at breaks prone to undergo repair via homologous recombination (HR). Time-resolved experiments revealed that these loops form during HR, much later than repair foci formation, but coincident with end resection and RAD51 assembly. RAD51 showed two-tiered accumulation around Cas9 breaks, with a broad (~Mb) domain arising from the homology search, the process by which the broken DNA ends scan the genome in search of an HR donor. The broad domain is regulated by cohesin unloader and is constrained by topologically associated domain (TAD) boundaries, and it overlaps with the chromatin regions that are reeled in through the break-anchored loop, suggesting a role for cohesin loop extrusion in the homology search. In support of this hypothesis, depletion of the cohesin subunit Nipbl results in reduced HR efficiency, and this effect is more pronounced when the HR donor is within the same TAD but positioned hundreds of kilobases from the break site. Our data indicates that loop-extruding cohesin promotes the mammalian homology search by promoting break-chromatin interactions within the damaged TAD.