Models for the architecture of the human inner kinetochore CCAN complex on centromeric α-satellite CENP-A nucleosome arrays

Models for the architecture of the human inner kinetochore CCAN complex on centromeric α-satellite CENP-A nucleosome arrays | 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 Models for the architecture of the human inner kinetochore CCAN complex on centromeric α-satellite CENP-A nucleosome arrays David Barford, Cong Yu, Kyle Muir, Jing Yang, Ziguo Zhang, Stephen McLaughlin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8029672/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 Human kinetochores assemble onto centromeric DNA defined by tandem copies of thousands of the 171 bp α-satellite repeat sequence. The centromere-specific CENP-A nucleosome (CENP-A Nuc ) recruits the inner kinetochore constitutive centromere associated network (CCAN) complex. A previous cryo-EM structure of CCAN bound to a CENP-A Nuc reconstituted with a 171 bp α-satellite repeat sequence showed how extranucleosomal linker DNA threads through an internal tunnel in the CCAN complex. To understand the higher-order architecture of the inner kinetochore assembled onto α-satellite repeat arrays we have determined cryo-EM structures of CCAN with longer DNA sequences. These include free DNA and single and dimeric α-satellite repeats with CENP-A nucleosomes. We show from the structures of CCAN bound to both free DNA and monomeric CENP-A Nuc that CCAN engages 65-70 bp of DNA comprising 30-35 bp of an upstream α-satellite repeat. This upstream DNA interacts with the histone fold domain subunits of the CENP-TWSX module in a manner resembling how DNA is wrapped in nucleosomes. A complex of CCAN assembled onto a dimeric α-satellite repeat with two CENP-A nucleosomes showed that CCAN can only be accommodated on the linker DNA by unwrapping DNA from the CENP-TWSX module together with 25 bp of DNA from the upstream nucleosome. We discuss the implications of these results for models of CCAN assembly on arrays of α-satellite chromatin containing CENP-A Nuc . Biological sciences/Biochemistry/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Cell biology/Chromosomes/Kinetochores Biological sciences/Cell biology/Chromosomes/Centromeres Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryVideo1.mp4 Supplementary Video1 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. 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