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bioRxiv · 10.1101/2025.11.04.686567

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

Yu, C.·Muir, K. W.·Yang, J.·Zhang, Z.·McLaughlin, S. H.·Barford, D.
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Abstract

Human kinetochores assemble onto centromeric DNA defined by tandem copies of thousands of the 171 bp -satellite repeat sequence. The centromere-specific CENP-A nucleosome (CENP-ANuc) recruits the inner kinetochore constitutive centromere associated network (CCAN) complex. A previous cryo-EM structure of CCAN bound to a CENP-ANuc reconstituted with a 171 bp -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 -satellite repeat arrays, we have determined cryo-EM structures of CCAN with longer DNA sequences. These include free DNA and single and dimeric -satellite repeats with CENP-A nucleosomes. We show from the structures of CCAN bound to both free DNA and monomeric CENP-ANuc that CCAN engages 65-70 bp of DNA comprising 30-35 bp of an upstream -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 -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 -satellite chromatin containing CENP-ANuc.

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Yu, C., Muir, K. W., Yang, J., Zhang, Z., McLaughlin, S. H., Barford, D.. 2025-11-04. Models for the architecture of the human inner kinetochore CCAN complex on centromeric α-satellite CENP-A nucleosome arrays. https://doi.org/10.1101/2025.11.04.686567

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