RAD51 D-loop structures reveal the mechanism of eukaryotic RAD51-mediated strand exchange

RAD51 D-loop structures reveal the mechanism of eukaryotic RAD51-mediated strand exchange | 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 RAD51 D-loop structures reveal the mechanism of eukaryotic RAD51-mediated strand exchange Meng-Chiao Ho, Shih-Chi Luo, Cheng-Han Yang, Hsin-Yi Yeh, Cheng-Wei Lin, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6511672/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Strand exchange is a key step in homologous recombination, enabling template-based repair of DNA double-strand breaks. Eukaryotic RAD51 forms an ATP-dependent helical presynaptic filament on single-stranded DNA (ssDNA), which then searches for homologous double-stranded DNA (dsDNA), and catalyzes the strand exchange to form a D-loop in an ATP hydrolysis-independent manner. The molecular mechanism by which RAD51 facilitates dsDNA unwinding and pairing remains unclear. Here, we present cryo-EM structures of RAD51 minifilaments bound to homologous dsDNA, capturing five intermediates from dsDNA recruitment to D-loop propagation. These structures, together with molecular dynamics simulations, suggest a stepwise mechanism: the conserved N-terminal domain (NTD) recruits and bends the dsDNA, weakening base pairing near the exchange site. Subsequent engagement with positively-charged regions, including the loop L2 and loop Arg303–Arg306, further bends the homologous dsDNA, thereby not only positioning it closer to the strand exchange site but also inducing local base-pair opening. Additionally, the loop L2 (Met278 and Phe279) inserts between strands, and the secondary DNA binding sites (S2 sites) capture the displaced strand to prevent strand reannealing. Together, our findings provide detailed insight into a spatially coordinated mechanism of strand exchange by RAD51. Biological sciences/Biochemistry/DNA Biological sciences/Biochemistry/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Molecular biology/DNA recombination Full Text Additional Declarations There is NO Competing Interest. Supplementary Files MovieS1.pptx movieS1 MovieS2.pptx movieS2 RAD51supplementaryFinalFinal.pdf Supplementary figures and tables NCOMMS2530959TsharpenMAPPDBs.zip Map and coordinates files Cite Share Download PDF Status: Published Journal Publication published 01 Dec, 2025 Read the published version in Nature Communications → 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6511672","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":448459237,"identity":"fe424b0d-7738-4abf-93a2-bb55aa8baf88","order_by":0,"name":"Meng-Chiao 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