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

NHEJ-mediated DNA synapsis and RPA-driven MRX exonuclease balance DNA end resection to control DSB repair pathway choice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article NHEJ-mediated DNA synapsis and RPA-driven MRX exonuclease balance DNA end resection to control DSB repair pathway choice Petr Cejka, Stefan Braunshier, Elda Cannavo, Ananya Acharya, Giordano Reginato, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8862494/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version 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 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. Biological sciences/Biochemistry/DNA Biological sciences/Biochemistry/Enzyme mechanisms Full Text Additional Declarations There is NO Competing Interest. Supplementary Files NHEJRPAExtendedData.pdf Extended data Figures and Tables Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8862494","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":597048672,"identity":"bc0bbfa8-4656-41a9-bdd7-1467a8be76b3","order_by":0,"name":"Petr 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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. 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