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

Abstract

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 resection, 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 find that MRX-pSae2 is inhibited structurally. DNA synapsis, a dynamic NHEJ intermediate that bridges DSBs, strongly suppresses resection. Non-ligatable ends are 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 inhibit 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.

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BibTeX RIS

BibTeX

@article{braunshier2026nhejmediated,
  title = {{NHEJ-mediated DNA synapsis and RPA-driven MRX exonuclease balance DNA end resection to control DSB repair}},
  author = {Stefan Braunshier and Elda Cannavo and Vincent Morin and Sriram KK and Ananya Acharya and Giordano Reginato and Florian Morati and Virginie Ropars and Raphael Guerois and Jean-Baptiste Charbonnier and Fredrik Westerlund and Terence Strick and Petr Cejka},
  journal = {Nature Communications},
  year = {2026},
  month = oct,
  doi = {10.1038/s41467-026-78670-y},
  url = {https://www.nature.com/articles/s41467-026-78670-y},
  note = {CC BY 4.0},
}

RIS

TY  - JOUR
TI  - NHEJ-mediated DNA synapsis and RPA-driven MRX exonuclease balance DNA end resection to control DSB repair
AU  - Stefan Braunshier
AU  - Elda Cannavo
AU  - Vincent Morin
AU  - Sriram KK
AU  - Ananya Acharya
AU  - Giordano Reginato
AU  - Florian Morati
AU  - Virginie Ropars
AU  - Raphael Guerois
AU  - Jean-Baptiste Charbonnier
AU  - Fredrik Westerlund
AU  - Terence Strick
AU  - Petr Cejka
PY  - 2026
DA  - 2026/10/22
JO  - Nature Communications
DO  - 10.1038/s41467-026-78670-y
UR  - https://www.nature.com/articles/s41467-026-78670-y
AB  - 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 resection, 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 find that MRX-pSae2 is inhibited structurally. DNA synapsis, a dynamic NHEJ intermediate that bridges DSBs, strongly suppresses resection. Non-ligatable ends are 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 inhibit 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.
SN  - 2041-1723
ER  -

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