NHEJ-mediated DNA synapsis and RPA-driven MRX exonuclease balance DNA end resection to control DSB repair pathway choice

DNA double-strand breaks (DSBs) are repaired by Ku-dependent non-homologous end-joining (NHEJ) or DNA end resection-dependent homologous recombination (HR). Previously, it was shown that short-range resection by MRX-pSae2 can remove Ku from DSBs and initiate resec-tion, showing flexibility in DSB repair pathway choice. However, which NHEJ intermediates can be disrupted by resection factors remains unknown. Using reconstituted reactions with purified S. cerevisiae proteins, we show here that short-range resection by MRX-pSae2 is not directly inhibited by any of the core NHEJ factors. Instead, we found that MRX-pSae2 is inhibited structurally. DNA synapsis, a dynamic NHEJ intermediate that bridges DSBs, strongly suppresses resection. Non-ligatable ends were refractory to MRX-pSae2 once synapsis formed, and NHEJ assemblies with catalytic mutants of DNA ligase IV that permit synapsis but do not allow ligation likewise inhibited resection. We further demonstrate that removal of Ku from DSBs requires RPA, which interacts with MRX and pSae2 to synergistically stimulate MRX 3′-5′ exonuclease activity, generating free 3′ overhangs. Our data support a model in which DNA synapsis temporarily prevents resection, allowing NHEJ to execute ligation. If ligation fails and synapsis dissociates, MRX-pSae2 and RPA promote resection that removes non-productive NHEJ assemblies and channels DSB repair toward HR. The data help explain the suppression of NHEJ by RPA observed in previous genetic experiments.