Abstract Accurate chromosome segregation, controlled by kinetochore-mediated chromatid attachments to the mitotic spindle, ensures the faithful inheritance of genetic information. Kinetochores assemble onto specialized CENP-A nucleosomes (CENP-A Nuc ) of centromeric chromatin. In humans, this is mostly organized as thousands of copies of an ∼171 bp α -satellite repeat. Here, we describe the cryo-EM structure of the human inner kinetochore CCAN (Constitutive Centromere Associated Network) complex bound to CENP-A Nuc reconstituted onto α-satellite DNA. CCAN forms edge-on contacts with CENP-A Nuc , while a linker DNA segment of the α-satellite repeat emerges from the fully-wrapped end of the nucleosome to thread through the central CENP-LN channel which tightly grips the DNA. The CENP-TWSX histone-fold module, together with CENP-HIK Head , further augments DNA binding and partially wraps the linker DNA in a manner reminiscent of canonical nucleosomes. Our study suggests that the topological entrapment of the α -satellite repeat linker DNA by CCAN provides a robust mechanism by which the kinetochore withstands the pushing and pulling of centromeres associated with chromosome congression and segregation forces. One-Sentence Summary The human inner kinetochore CCAN complex tightly grips the linker DNA of the α-satellite CENP-A nucleosome.

Structure of the human inner kinetochore bound to a centromeric CENP-A nucleosome