The BRCT domain enhances DNA binding and catalytic efficiency of fungal PARPs

The BRCT domain enhances DNA binding and catalytic efficiency of fungal PARPs | 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 The BRCT domain enhances DNA binding and catalytic efficiency of fungal PARPs Johannes Rack, Adam Bainbridge, Tiberiu-Marius Gianga, Rohanah Hussain, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7860866/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 Aspergillus fumigatus infections are a major yet often neglected global health challenge magnified by a growing at-risk population, limited treatment options, and the emergence of drug-resistant strains. Regulation of the DNA damage response (DDR) by ADP-ribosylation signalling has recently emerged as an important feature of fungal pathogenesis, but the underlying mechanisms remained largely elusive. Here we present a comprehensive phylogenetic and functional characterisation of Af -PARP1, the A. fumigatus PARP homologue. Our data reveal Af -PARP1 as a DNA-dependent poly(ADP-ribosyl)transferase with unique domain architecture, DNA damage selectivity, and activation dynamics distinct from its mammalian and plant homologues. We show that the fungal specific BRCT domain plays a crucial role in both damage recognition and ADP-ribosylation signal establishment. Collectively, our findings reveal a divergence in DDR-associated ADP-ribosylation specific to fungi, highlighting the potential of this signalling pathway as target for antifungal therapy. Biological sciences/Biochemistry/Enzyme mechanisms Biological sciences/Structural biology/SAXS Biological sciences/Biochemistry/DNA Full Text Additional Declarations There is NO Competing Interest. Supplementary Files AGBAfPARP1BRCTSupplementaryInformation.pdf Manuscript Supplementary Information ExtendedDataFigure3.pdf Extended Data Figure 3. Conservation of BRCT domain within Fungi . ExtendedDataFigure6.pdf Extended Data Figure 6. PARP-derived BRCT domain show little conservation between homologues. ExtendedDataFigure4.pdf Extended Data Figure 4. SAXS quality parameters and CD near-UV spectra for Af -PARP1 and its DNA complexes. ExtendedDataFigure5.pdf Extended Data Figure 5. The DNA surface of the BRCT domain has a positive surface potential. ExtendedDataFigure1.pdf Extended Data Figure 1. Activation of animal and plant PARPs by 5’-phosphorylated DNA damage mimetics. ExtendedDataFigure2.pdf Extended Data Figure 2. DNA damage recognition by the Af -PARP1 WGR domain. 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-7860866","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":530585168,"identity":"d903fb46-daf0-45a6-bd05-b0cff473dd24","order_by":0,"name":"Johannes Rack","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYBACxgYQecAGWYyNKC1pQIKZSC0QcOAwCVqYG3gffvhx5rycwfH+g48rGOzkGSTSEgg4jN1YsufGbSBxmNnwDEOyYYNE2gECWtgYpBk+3E7sl0hmk2xgYE5gkEhvIKSF+TfDh3P1bfKP2X82MNQTpYVNmuHGgQR+CWY2YPAdBmoh5LBmNjbLnjPJhjN7ko0lGwyOG7bxPEvAq8WwvY35xo9jdvIGxw8+/NhQUS3Pz55mgF9LMwrXgIiIlCekYBSMglEwCkYBAwCVQj3wH1HFpAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-8341-6439","institution":"University of Exeter","correspondingAuthor":true,"prefix":"","firstName":"Johannes","middleName":"","lastName":"Rack","suffix":""},{"id":530585169,"identity":"abfc8338-18d6-46f3-ab1f-af3e51fbe48e","order_by":1,"name":"Adam Bainbridge","email":"","orcid":"","institution":"University of Exeter","correspondingAuthor":false,"prefix":"","firstName":"Adam","middleName":"","lastName":"Bainbridge","suffix":""},{"id":530585170,"identity":"f3bf000d-9e09-439c-9cae-dfd782afa06a","order_by":2,"name":"Tiberiu-Marius Gianga","email":"","orcid":"","institution":"Diamond Light Source Ltd","correspondingAuthor":false,"prefix":"","firstName":"Tiberiu-Marius","middleName":"","lastName":"Gianga","suffix":""},{"id":530585171,"identity":"c91cf2bf-8a05-4d43-9f35-cf67132396a4","order_by":3,"name":"Rohanah Hussain","email":"","orcid":"https://orcid.org/0000-0001-6207-6631","institution":"Diamond Light Source","correspondingAuthor":false,"prefix":"","firstName":"Rohanah","middleName":"","lastName":"Hussain","suffix":""},{"id":530585172,"identity":"201bc554-8d69-4527-a8a3-d052e1155150","order_by":4,"name":"Nicolas Helmstetter","email":"","orcid":"","institution":"University of Exeter","correspondingAuthor":false,"prefix":"","firstName":"Nicolas","middleName":"","lastName":"Helmstetter","suffix":""},{"id":530585173,"identity":"9736280b-d2b9-4ff1-80d8-6054c78de12d","order_by":5,"name":"Rhys Farrer","email":"","orcid":"","institution":"University of Exeter","correspondingAuthor":false,"prefix":"","firstName":"Rhys","middleName":"","lastName":"Farrer","suffix":""},{"id":530585174,"identity":"1fdb9266-853d-4db5-a213-62547d11dfe7","order_by":6,"name":"Giuliano Siligardi","email":"","orcid":"https://orcid.org/0000-0002-4667-6423","institution":"B23 Beamline, Diamond Light Source, Harwell Science Innovation Campus","correspondingAuthor":false,"prefix":"","firstName":"Giuliano","middleName":"","lastName":"Siligardi","suffix":""}],"badges":[],"createdAt":"2025-10-14 17:05:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7860866/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7860866/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":94990367,"identity":"5c8dbe89-ffd6-44b3-9ca2-619f2a6f9be2","added_by":"auto","created_at":"2025-11-03 07:16:38","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5020669,"visible":true,"origin":"","legend":"","description":"","filename":"AGBAfPARP1BRCTMainManuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7860866/v1_covered_8ec27959-9a9a-4f2b-8428-96a600f6a13b.pdf"},{"id":94985296,"identity":"e6d65301-7f58-4391-823a-50668f7da105","added_by":"auto","created_at":"2025-11-03 06:57:52","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11304674,"visible":true,"origin":"","legend":"Manuscript Supplementary Information","description":"","filename":"AGBAfPARP1BRCTSupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7860866/v1/4befe27ed2acb77db1d543e8.pdf"},{"id":94840318,"identity":"57bf1791-4e35-4313-95c6-872a6652e2df","added_by":"auto","created_at":"2025-10-31 09:15:52","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":817148,"visible":true,"origin":"","legend":"Extended Data Figure 3. Conservation of BRCT domain within \u003ci\u003eFungi\u003c/i\u003e.","description":"","filename":"ExtendedDataFigure3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7860866/v1/8895da24c9daa1eb8010324f.pdf"},{"id":94840314,"identity":"22b93deb-3b15-41a5-8c29-99f4aaac89db","added_by":"auto","created_at":"2025-10-31 09:15:52","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1223560,"visible":true,"origin":"","legend":"\u003cp\u003eExtended Data Figure 6. PARP-derived BRCT domain show little conservation between homologues.\u003c/p\u003e","description":"","filename":"ExtendedDataFigure6.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7860866/v1/ece255dce653b56020ef176b.pdf"},{"id":94840336,"identity":"6d590dbe-8904-4396-bfa4-70db0dbdaa5a","added_by":"auto","created_at":"2025-10-31 09:15:53","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":2261508,"visible":true,"origin":"","legend":"\u003cp\u003eExtended Data Figure 4. SAXS quality parameters and CD near-UV spectra for \u003cem\u003eAf\u003c/em\u003e-PARP1 and its DNA complexes.\u003c/p\u003e","description":"","filename":"ExtendedDataFigure4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7860866/v1/61b8fa506ff64f61d5ba800e.pdf"},{"id":94840330,"identity":"617c9905-2012-4024-9995-0fd9bfdce6f7","added_by":"auto","created_at":"2025-10-31 09:15:52","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":7466865,"visible":true,"origin":"","legend":"\u003cp\u003eExtended Data Figure 5. The DNA surface of the BRCT domain has a positive surface potential.\u003c/p\u003e","description":"","filename":"ExtendedDataFigure5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7860866/v1/3458826525b0b2325cceca41.pdf"},{"id":94840332,"identity":"a259a710-b17b-47d8-9174-bc06b33b161a","added_by":"auto","created_at":"2025-10-31 09:15:53","extension":"pdf","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":10299421,"visible":true,"origin":"","legend":"Extended Data Figure 1. Activation of animal and plant PARPs by 5\u0026#x2019;-phosphorylated DNA damage mimetics.","description":"","filename":"ExtendedDataFigure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7860866/v1/a625a24c159605156bb66707.pdf"},{"id":94840329,"identity":"ab2fe76d-26c5-4bd5-8dda-a681d74ceb78","added_by":"auto","created_at":"2025-10-31 09:15:52","extension":"pdf","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":12971185,"visible":true,"origin":"","legend":"Extended Data Figure 2. DNA damage recognition by the \u003ci\u003eAf\u003c/i\u003e-PARP1 WGR domain.","description":"","filename":"ExtendedDataFigure2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7860866/v1/ea567d50d1f68b3708eba62f.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"The BRCT domain enhances DNA binding and catalytic efficiency of fungal PARPs","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-7860866/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7860866/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ci\u003eAspergillus fumigatus\u003c/i\u003e infections are a major yet often neglected global health challenge magnified by a growing at-risk population, limited treatment options, and the emergence of drug-resistant strains. Regulation of the DNA damage response (DDR) by ADP-ribosylation signalling has recently emerged as an important feature of fungal pathogenesis, but the underlying mechanisms remained largely elusive. Here we present a comprehensive phylogenetic and functional characterisation of \u003ci\u003eAf\u003c/i\u003e-PARP1, the \u003ci\u003eA. fumigatus\u003c/i\u003e PARP homologue. Our data reveal \u003ci\u003eAf\u003c/i\u003e-PARP1 as a DNA-dependent poly(ADP-ribosyl)transferase with unique domain architecture, DNA damage selectivity, and activation dynamics distinct from its mammalian and plant homologues. We show that the fungal specific BRCT domain plays a crucial role in both damage recognition and ADP-ribosylation signal establishment. 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