Abstract Human kinetochores assemble onto centromeric DNA comprising repetitive arrays of the 171 bp α-satellite sequence. To understand the higher-order architecture of the inner kinetochore assembled onto α-satellite arrays, we show cryo-EM structures of CCAN with free DNA, and α-satellite repeat monomers and dimers with CENP-A nucleosomes. CCAN bound to free DNA and a monomeric CENP-A nucleosome engages 70 bp of DNA comprising 30 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 nucleosomes wrap DNA gyres. A complex of CCAN assembled onto a dimeric α-satellite repeat with two CENP-A nucleosomes shows that CCAN can only be accommodated on the linker DNA by unwrapping DNA from both the CENP-TWSX module and the upstream nucleosome. We discuss the implications of these results for models of CCAN assembly on arrays of α-satellite chromatin containing CENP-A nucleosomes.
Yu et al. (Tue,) studied this question.