Structural basis of Ku-mediated activation of WRN exonuclease activity

Structural basis of Ku-mediated activation of WRN exonuclease activity | Research Square 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Article Structural basis of Ku-mediated activation of WRN exonuclease activity Virginie Ropars, Sayma Zahid, Jeanne Chauvat, Ilaria Ceppi, Philippe Frit, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5965979/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 Werner (WRN) is the only human RecQ helicase family member with DNA exonuclease activity. WRN promotes genome stability through its functions in DNA replication, repair and telomere maintenance, the deficiency of which presents clinically as Werner syndrome, causing premature aging and cancer predisposition. The main DNA double strand-break sensor Ku70/80 heterodimer (Ku) is a known partner of WRN, which stimulates its nuclease activity. However, the molecular basis of Ku-WRN interplay is currently unknown. Here, we present a high resolution cryo-EM structure of human Ku bound to DNA in complex with the N-terminal WRN exonuclease domain. This structure reveals multiple interaction sites between WRN and the Ku:DNA complex. The catalytic domain of WRN-exo engages with the DNA ends, stabilized by the vWA-like Ku80 domain interacting with the N-terminal APLF-like Ku binding motif (A-KBM) of WRN. Most surprisingly, we visualize the SAP domain of Ku70 stabilized within this complex, and we identify specific contacts mediating this interaction. These interactions were validated by assessing the impact of point mutations on either side of the Ku-WRN interfaces on exonuclease activity with purified recombinant proteins, and on live protein recruitment at biphoton laser-damaged nuclear sites. Finally, we show that disruption of WRN-Ku70 interaction results in aberrant resection of stalled replication forks. Together, we define the architecture of the Ku-WRN exonuclease domain interface and its impact on WRN exonuclease activity, recruitment and replication fork processing. Biological sciences/Molecular biology/DNA replication/Stalled forks Biological sciences/Genetics/Genomic instability Biological sciences/Molecular biology/DNA damage and repair/Double-strand DNA breaks Biological sciences/Biochemistry/Structural biology/Electron microscopy/Cryoelectron microscopy Full Text Additional Declarations There is NO Competing Interest. Supplementary Files D1292144072valreportfullP1v2.pdf PDB validation report file ReportingSummary.pdf Reporting Summary SourceData.xlsx Source 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 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

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