Real-Time Visualization of G2L4 Reverse Transcriptase in DNA Repair via Microhomology-Mediated End Joining

Real-Time Visualization of G2L4 Reverse Transcriptase in DNA Repair via Microhomology-Mediated End Joining | 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 Real-Time Visualization of G2L4 Reverse Transcriptase in DNA Repair via Microhomology-Mediated End Joining Yi-Chih Lin, Pangmiaomiao Zhang, Mo Guo, Y. Jessie Zhang, Alan Lambowitz This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8780377/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 Double-strand break repair (DSBR) is essential for genome integrity, yet mechanistic details of error-prone microhomology-mediated end joining (MMEJ) remain unclear. A bacterial group II intron-like reverse transcriptase, G2L4 RT, has been implicated in MMEJ, but how it executes DSBR is unknown. Using high-speed atomic force microscopy (HS-AFM), we directly visualize G2L4 RT-mediated DSBR via MMEJ. We observe that G2L4 RT dimers exhibit RT3a plug protrusion upon DNA engagement and catalyze MMEJ by binding and stabilizing a 4-bp annealed microhomology and filling adjacent single-strand gaps with dNTPs. We also observe Mn 2+ -stimulated terminal transferase activity that generates elongated and branched DNA intermediates prior to ligation. With T4 DNA ligase, we visualize binding near nick sites and real-time nick sealing, which stabilizes the repaired products and suppresses off-pathway branching. These results reveal how G2L4 RT and ligase activities shape MMEJ intermediates and outcomes. Biological sciences/Biophysics/Single-molecule biophysics Biological sciences/Molecular biology/DNA damage and repair/Non-homologous-end joining Biological sciences/Biochemistry/Structural biology/Atomic force microscopy Full Text Additional Declarations There is NO Competing Interest. Supplementary Files MovieS1StructuraldynamicsofAPOG2L4RTdimer.avi Supplementary Movie S1. Structural dynamics of APO-G2L4 RT dimer MovieS2StructuraldynamicsofanotherAPOG2L4RTdimer.avi Supplementary Movie S2. Structural dynamics of another APO-G2L4 RT dimer MovieS3DissociationofaG2L4dimerinMn2condition.avi Supplementary Movie S3. Dissociation of a G2L4 dimer in Mn 2+ condition MovieS4StructuraldynamicsofindividualMMEJsubstrates.avi Supplementary Movie S4. Structural dynamics of individual MMEJ substrates MovieS5StructuraldynamicsofaMMEJdimer.avi Supplementary Movie S5. Structural dynamics of a MMEJ dimer MovieS6AssociationanddissociationofaMMEJdimer.avi Supplementary Movie S6. Association and dissociation of a MMEJ dimer MovieS7AMMEJdimerclampedbyaG2L4RTdimer.avi Supplementary Movie S7. A MMEJ dimer clamped by a G2L4 RT dimer MovieS8G2L4RTdimerwithaRT3aplugprotrusionuponMMEJsubstrateengagement.avi Supplementary Movie S8. G2L4 RT dimer with a RT3a plug protrusion upon MMEJ substrate engagement MovieS9G2L4RTdimerrepositionsonaMMEJdimer.avi Supplementary Movie S9. G2L4 RT dimer repositions on a MMEJ dimer MovieS10MolecularinteractionsbetweenG2L4RTandlongDNAproducts.avi Supplementary Movie S10. Molecular interactions between G2L4 RT and long DNA products MovieS11SelfrearrangementsoftheDNAbackboneatanickedsitetoformabranchedmorphology.avi Supplementary Movie S11. Self-rearrangements of the DNA backbone at a nicked site to form a branched morphology MovieS12T4DNAligaserepairsaMMEJdimer.avi Supplementary Movie S12. T4 DNA ligase repairs a MMEJ dimer 20260201G2L4inMMEJrepairSIv20.pdf Supporting Information. 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-8780377","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":603039972,"identity":"b63b3d59-8237-45aa-b96d-e0da242b11ce","order_by":0,"name":"Yi-Chih Lin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYDACZgY2hsQGBhk2BuYDcEEJYrTwsDGwJTDANOHXwgDUwgjUwsDAY0CcFoPjzM8ePNzBwMMn3fNN+kPFYXn5BuaDt3nwaTnMZm6QeAboMJmz2yQOnEkz3HCALdkavxYeNonENqAWidxtEgfbbBg3MPCYSROpJeeZxMF/EvbzG/i/Ea2FTeJgg01iwwEeNrxaJA+zmQG1SAC1pBlbnDmWlrzhMJux5Rw8WvjOH34m+bPNRk5+RvLDGxU1h23ntzc/vPEGjxaFA2AKOSKY8SgHAfkGAgpGwSgYBaNgFDAAAJueRFxGQ/HjAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-6498-215X","institution":"The University of Texas at Austin","correspondingAuthor":true,"prefix":"","firstName":"Yi-Chih","middleName":"","lastName":"Lin","suffix":""},{"id":603039973,"identity":"3e589dcc-1038-49a0-9147-4d55873ae6f4","order_by":1,"name":"Pangmiaomiao Zhang","email":"","orcid":"https://orcid.org/0000-0002-9701-4841","institution":"The University of Texas at Austin","correspondingAuthor":false,"prefix":"","firstName":"Pangmiaomiao","middleName":"","lastName":"Zhang","suffix":""},{"id":603039974,"identity":"17a0b097-2d5f-4545-a2e0-93dc69b9d121","order_by":2,"name":"Mo Guo","email":"","orcid":"https://orcid.org/0009-0000-9103-975X","institution":"The University of Texas at Austin","correspondingAuthor":false,"prefix":"","firstName":"Mo","middleName":"","lastName":"Guo","suffix":""},{"id":603039975,"identity":"9a05e70a-88da-498b-841e-48e7881c6518","order_by":3,"name":"Y. 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Structural dynamics of individual MMEJ substrates","description":"","filename":"MovieS4StructuraldynamicsofindividualMMEJsubstrates.avi","url":"https://assets-eu.researchsquare.com/files/rs-8780377/v1/0c5a806127499c53cefaa0f6.avi"},{"id":104573233,"identity":"39943c27-36b7-4500-bc09-d408fbab7add","added_by":"auto","created_at":"2026-03-13 13:12:41","extension":"avi","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1201434,"visible":true,"origin":"","legend":"Supplementary Movie S5. Structural dynamics of a MMEJ dimer","description":"","filename":"MovieS5StructuraldynamicsofaMMEJdimer.avi","url":"https://assets-eu.researchsquare.com/files/rs-8780377/v1/9b9b36b0453ca9de49339e08.avi"},{"id":104573232,"identity":"13c9b14e-581f-4305-bfbb-9e86cb865622","added_by":"auto","created_at":"2026-03-13 13:12:41","extension":"avi","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1233244,"visible":true,"origin":"","legend":"Supplementary Movie S6. Association and dissociation of a MMEJ dimer","description":"","filename":"MovieS6AssociationanddissociationofaMMEJdimer.avi","url":"https://assets-eu.researchsquare.com/files/rs-8780377/v1/9f11e89792cc405c94dee185.avi"},{"id":104573238,"identity":"5de39946-3478-4824-bdd9-85ec20c253eb","added_by":"auto","created_at":"2026-03-13 13:12:41","extension":"avi","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":15262026,"visible":true,"origin":"","legend":"Supplementary Movie S7. A MMEJ dimer clamped by a G2L4 RT dimer","description":"","filename":"MovieS7AMMEJdimerclampedbyaG2L4RTdimer.avi","url":"https://assets-eu.researchsquare.com/files/rs-8780377/v1/37d4512e68f32ca4a6184a67.avi"},{"id":104781029,"identity":"612a9937-e6bf-4d20-97d2-07331c4aab9c","added_by":"auto","created_at":"2026-03-17 07:54:30","extension":"avi","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":871910,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Movie S8. G2L4 RT dimer with a RT3a plug protrusion upon MMEJ substrate engagement\u003c/p\u003e","description":"","filename":"MovieS8G2L4RTdimerwithaRT3aplugprotrusionuponMMEJsubstrateengagement.avi","url":"https://assets-eu.researchsquare.com/files/rs-8780377/v1/c750acb71f96a4475fd7a362.avi"},{"id":104573240,"identity":"9e3f6c83-2156-4dba-b485-e622b3f86429","added_by":"auto","created_at":"2026-03-13 13:12:41","extension":"avi","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":9804318,"visible":true,"origin":"","legend":"Supplementary Movie S9. G2L4 RT dimer repositions on a MMEJ dimer","description":"","filename":"MovieS9G2L4RTdimerrepositionsonaMMEJdimer.avi","url":"https://assets-eu.researchsquare.com/files/rs-8780377/v1/cbeb125a0b7c6824d27acd74.avi"},{"id":104573235,"identity":"84cf9dca-535a-4b8d-a942-7ace8cb3fd4a","added_by":"auto","created_at":"2026-03-13 13:12:41","extension":"avi","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":9120280,"visible":true,"origin":"","legend":"Supplementary Movie S10. Molecular interactions between G2L4 RT and long DNA products","description":"","filename":"MovieS10MolecularinteractionsbetweenG2L4RTandlongDNAproducts.avi","url":"https://assets-eu.researchsquare.com/files/rs-8780377/v1/7a3a8653509f624be5be0173.avi"},{"id":104781580,"identity":"5f1b3293-b10e-41ce-b4f2-938a71e4fc8f","added_by":"auto","created_at":"2026-03-17 07:55:57","extension":"avi","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":13786416,"visible":true,"origin":"","legend":"Supplementary Movie S11. Self-rearrangements of the DNA backbone at a nicked site to form a branched morphology","description":"","filename":"MovieS11SelfrearrangementsoftheDNAbackboneatanickedsitetoformabranchedmorphology.avi","url":"https://assets-eu.researchsquare.com/files/rs-8780377/v1/150b355a485f45d320d90b6c.avi"},{"id":104573236,"identity":"847b7e5f-a09a-4401-bb03-df8344dc8c10","added_by":"auto","created_at":"2026-03-13 13:12:41","extension":"avi","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":6346038,"visible":true,"origin":"","legend":"Supplementary Movie S12. T4 DNA ligase repairs a MMEJ dimer","description":"","filename":"MovieS12T4DNAligaserepairsaMMEJdimer.avi","url":"https://assets-eu.researchsquare.com/files/rs-8780377/v1/780084905635a78090eae184.avi"},{"id":104573239,"identity":"ef1c4a74-b21c-479e-b4e0-2ac75ce23907","added_by":"auto","created_at":"2026-03-13 13:12:41","extension":"pdf","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":7092119,"visible":true,"origin":"","legend":"\u003cp\u003eSupporting Information.\u003c/p\u003e","description":"","filename":"20260201G2L4inMMEJrepairSIv20.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8780377/v1/fad961c5a76ecfcd4846a69b.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Real-Time Visualization of G2L4 Reverse Transcriptase in DNA Repair via Microhomology-Mediated End Joining","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-8780377/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8780377/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Double-strand break repair (DSBR) is essential for genome integrity, yet mechanistic details of error-prone microhomology-mediated end joining (MMEJ) remain unclear. A bacterial group II intron-like reverse transcriptase, G2L4 RT, has been implicated in MMEJ, but how it executes DSBR is unknown. Using high-speed atomic force microscopy (HS-AFM), we directly visualize G2L4 RT-mediated DSBR via MMEJ. We observe that G2L4 RT dimers exhibit RT3a plug protrusion upon DNA engagement and catalyze MMEJ by binding and stabilizing a 4-bp annealed microhomology and filling adjacent single-strand gaps with dNTPs. We also observe Mn\u003csup\u003e2+\u003c/sup\u003e-stimulated terminal transferase activity that generates elongated and branched DNA intermediates prior to ligation. With T4 DNA ligase, we visualize binding near nick sites and real-time nick sealing, which stabilizes the repaired products and suppresses off-pathway branching. These results reveal how G2L4 RT and ligase activities shape MMEJ intermediates and outcomes.","manuscriptTitle":"Real-Time Visualization of G2L4 Reverse Transcriptase in DNA Repair via Microhomology-Mediated End Joining","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-13 13:12:30","doi":"10.21203/rs.3.rs-8780377/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":"e07a9b90-e229-4e32-87e3-5a3e3687438c","owner":[],"postedDate":"March 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":64165622,"name":"Biological sciences/Biophysics/Single-molecule biophysics"},{"id":64165623,"name":"Biological sciences/Molecular biology/DNA damage and repair/Non-homologous-end joining"},{"id":64165624,"name":"Biological sciences/Biochemistry/Structural biology/Atomic force microscopy"}],"tags":[],"updatedAt":"2026-03-18T14:26:35+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-13 13:12:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8780377","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8780377","identity":"rs-8780377","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}