Authors
Yu, C., Muir, K. W., Yang, J., Zhang, Z., McLaughlin, S. H., Barford, D.
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.
Preprint server:
bioRxiv
The authors list and abstract were imported from bioRxiv on 06 Nov 2025.
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