Molecular basis for CSB stimulation of the SNM1A DNA repair nuclease

Molecular basis for CSB stimulation of the SNM1A DNA repair nuclease | 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 Molecular basis for CSB stimulation of the SNM1A DNA repair nuclease Peter McHugh, Rebecca Roddan, Matthew Schaich, Willlam Nathan, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7886236/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 The Cockayne Syndrome B (CSB, ERCC6) protein, interacts with the exonuclease SNM1A during transcription-coupled DNA interstrand (ICL) repair, with CSB facilitating localisation of SNM1A to ICL damage. The functional and mechanistic details of this interaction in DNA repair, however, have not been defined. Here, we demonstrate that CSB enhances SNM1A resection through ICLs and identify a specific interaction between the winged-helix domain of CSB and the nuclease core of SNM1A that is crucial for recruitment and enhancement of nuclease degradation. Biochemical and single-molecule studies on DNA containing sitespecific ICLs reveal that CSB increases the affinity of SNM1A to damaged DNA substrates and also alters the substrate conformation to enhance ICL processing by SNM1A. Notably, CSB was observed preferentially as a dimer when colocalised with SNM1A at ICLs, constrasting with its monomeric nature observed during repair initiation in classical transcription-coupled nucleotide excision repair. The combined results provide molecular insights into the basis of a direct contribution of CSB to a DNA repair reaction. Biological sciences/Chemical biology/Enzyme mechanisms Biological sciences/Biochemistry/Enzyme mechanisms Biological sciences/Molecular biology/DNA damage and repair Full Text Additional Declarations There is NO Competing Interest. Supplementary Files RoddanetalSI.pdf All supplemental data 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. 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-7886236","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":537892482,"identity":"cc3d08b2-e958-4e0e-b142-9e32afaa438f","order_by":0,"name":"Peter McHugh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYDADAwYGNoYPBkgiEjjVMiO0MM4gWQszD7IELi3m7P0HH1cwbJM3l0i/9timYFs0A3uPAcOPGobEmQ3YtVj2HGY2PMNw23DnjJxy4xyD27kNPMcSGHuOMSTOxmGLwY1kNskGhtsJBjdy0qTBWiSSDzDwNjAkzsOl5f5j9p9wLRZgLYkNjH/xabnBzMYI0ZJ+TJoBagszyBZcDrPsSTaWbDC4bbjhzBs2yR6gljagXw7LHJMwxuV9c/aDDz82VNyWNzie/kzix5/buf3sPYYP39TYyM44gMNhCJIHwmYD4gP4IhIputkf4FQ1CkbBKBgFIxsAAH8FWaKvi0GBAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-8679-4627","institution":"University of Oxford","correspondingAuthor":true,"prefix":"","firstName":"Peter","middleName":"","lastName":"McHugh","suffix":""},{"id":537892483,"identity":"e0752834-8249-4a55-87cf-4cefc182eeba","order_by":1,"name":"Rebecca Roddan","email":"","orcid":"https://orcid.org/0000-0002-9406-9011","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Rebecca","middleName":"","lastName":"Roddan","suffix":""},{"id":537892484,"identity":"98a5e651-c76d-4e7e-9664-9aa7d38fdefc","order_by":2,"name":"Matthew Schaich","email":"","orcid":"","institution":"University of Pittsburgh","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"","lastName":"Schaich","suffix":""},{"id":537892485,"identity":"314a56ff-a91b-47b2-a239-db176f886e06","order_by":3,"name":"Willlam Nathan","email":"","orcid":"","institution":"NCI, National Institutes of Health","correspondingAuthor":false,"prefix":"","firstName":"Willlam","middleName":"","lastName":"Nathan","suffix":""},{"id":537892486,"identity":"cb32f134-4817-4d2f-9d6b-0a56b77e2144","order_by":4,"name":"Iulia Kis","email":"","orcid":"https://orcid.org/0009-0000-1672-2532","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Iulia","middleName":"","lastName":"Kis","suffix":""},{"id":537892487,"identity":"78d2801b-fcc5-41e0-a936-ac91410b4b1e","order_by":5,"name":"Andrea Pierangelini","email":"","orcid":"https://orcid.org/0009-0009-9396-619X","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Pierangelini","suffix":""},{"id":537892488,"identity":"3ae900f9-290e-4965-9991-ff068e911b7c","order_by":6,"name":"Lucy Henderson","email":"","orcid":"https://orcid.org/0000-0002-0142-5444","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Lucy","middleName":"","lastName":"Henderson","suffix":""},{"id":537892489,"identity":"f029b4aa-ae8c-4375-a015-edb7fe7ef054","order_by":7,"name":"Raman van Wee","email":"","orcid":"https://orcid.org/0000-0001-8941-3693","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Raman","middleName":"van","lastName":"Wee","suffix":""},{"id":537892490,"identity":"bbe5555c-3b84-454f-9489-2af6e858d163","order_by":8,"name":"Louise Martin","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Louise","middleName":"","lastName":"Martin","suffix":""},{"id":537892491,"identity":"f0460129-5337-40a6-a000-b4dd33d3d52b","order_by":9,"name":"Afaf El-Sagheer","email":"","orcid":"","institution":"University of Southampton","correspondingAuthor":false,"prefix":"","firstName":"Afaf","middleName":"","lastName":"El-Sagheer","suffix":""},{"id":537892492,"identity":"94d75125-9c56-465c-90a8-6e2d190cb70d","order_by":10,"name":"Philipp Kukura","email":"","orcid":"https://orcid.org/0000-0003-0136-7704","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Philipp","middleName":"","lastName":"Kukura","suffix":""},{"id":537892493,"identity":"8d81fe99-22a3-48c5-911a-3d37140957c3","order_by":11,"name":"Tom Brown","email":"","orcid":"https://orcid.org/0000-0002-6538-3036","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Tom","middleName":"","lastName":"Brown","suffix":""},{"id":537892494,"identity":"4c82a7cd-0b0b-4550-badd-96ecef53fb4c","order_by":12,"name":"Darragh O’Brien","email":"","orcid":"https://orcid.org/0000-0003-4924-7795","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Darragh","middleName":"","lastName":"O’Brien","suffix":""},{"id":537892495,"identity":"6d5568fa-e46d-488c-afdc-029115bc8ddd","order_by":13,"name":"Joseph Newman","email":"","orcid":"","institution":"UNIVERSITY OF OXFORD","correspondingAuthor":false,"prefix":"","firstName":"Joseph","middleName":"","lastName":"Newman","suffix":""},{"id":537892496,"identity":"1b0da814-9bf1-4d6d-bde3-c42ad9b2660f","order_by":14,"name":"David Wilson iii","email":"","orcid":"","institution":"Hasselt University","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"Wilson","lastName":"iii","suffix":""},{"id":537892497,"identity":"0e63bc34-c031-4d6c-a6d2-d83b7b6a6769","order_by":15,"name":"Christopher Schofield","email":"","orcid":"https://orcid.org/0000-0002-0290-6565","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Christopher","middleName":"","lastName":"Schofield","suffix":""},{"id":537892498,"identity":"18e918a5-360c-448c-9bba-97f3b6aa506e","order_by":16,"name":"Bennett Houten","email":"","orcid":"https://orcid.org/0000-0002-4009-2478","institution":"University of Pittsburgh","correspondingAuthor":false,"prefix":"","firstName":"Bennett","middleName":"","lastName":"Houten","suffix":""}],"badges":[],"createdAt":"2025-10-17 11:35:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7886236/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7886236/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":96246585,"identity":"59c0055a-f0b8-4406-8c43-dfc7dbcef41f","added_by":"auto","created_at":"2025-11-19 07:26:17","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7548481,"visible":true,"origin":"","legend":"Article File","description":"","filename":"Roddanetalmain.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7886236/v1_covered_11d4a847-30cf-4b60-a0d1-a1f532d4599a.pdf"},{"id":96037969,"identity":"a328bbbf-bbd9-42e8-a66d-ad4efc6d54a3","added_by":"auto","created_at":"2025-11-17 03:32:32","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":30922239,"visible":true,"origin":"","legend":"All supplemental data","description":"","filename":"RoddanetalSI.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7886236/v1/243ba918c44646dd9583ff8c.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Molecular basis for CSB stimulation of the SNM1A DNA repair nuclease","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7886236/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7886236/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The Cockayne Syndrome B (CSB, ERCC6) protein, interacts with the exonuclease SNM1A\r\nduring transcription-coupled DNA interstrand (ICL) repair, with CSB facilitating localisation of\r\nSNM1A to ICL damage. The functional and mechanistic details of this interaction in DNA\r\nrepair, however, have not been defined. Here, we demonstrate that CSB enhances SNM1A\r\nresection through ICLs and identify a specific interaction between the winged-helix domain of\r\nCSB and the nuclease core of SNM1A that is crucial for recruitment and enhancement of\r\nnuclease degradation. Biochemical and single-molecule studies on DNA containing sitespecific\r\nICLs reveal that CSB increases the affinity of SNM1A to damaged DNA substrates\r\nand also alters the substrate conformation to enhance ICL processing by SNM1A. Notably,\r\nCSB was observed preferentially as a dimer when colocalised with SNM1A at ICLs,\r\nconstrasting with its monomeric nature observed during repair initiation in classical\r\ntranscription-coupled nucleotide excision repair. The combined results provide molecular\r\ninsights into the basis of a direct contribution of CSB to a DNA repair reaction.","manuscriptTitle":"Molecular basis for CSB stimulation of the SNM1A DNA repair nuclease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-17 03:32:27","doi":"10.21203/rs.3.rs-7886236/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4c6fa044-5e37-43a4-9185-afe40a288fe3","owner":[],"postedDate":"November 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":57207207,"name":"Biological sciences/Chemical biology/Enzyme mechanisms"},{"id":57207208,"name":"Biological sciences/Biochemistry/Enzyme mechanisms"},{"id":57207209,"name":"Biological sciences/Molecular biology/DNA damage and repair"}],"tags":[],"updatedAt":"2025-11-17T03:32:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-17 03:32:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7886236","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7886236","identity":"rs-7886236","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}