Structure of the inner kinetochore CCAN complex assembled onto a centromeric nucleosome.

Abstract

In eukaryotes, accurate chromosome segregation in mitosis and meiosis maintains genome stability and prevents aneuploidy. Kinetochores are large protein complexes, that by assembling onto specialized Cenp-A nucleosomes 1,2, function to connect centromeric chromatin to microtubules of the mitotic spindle 3,4. Whereas the centromeres of vertebrate chromosomes comprise Mb of DNA and attach to multiple microtubules, the simple point centromeres of budding yeast are connected to individual microtubules 5,6. All 16 budding yeast chromosomes assemble complete kinetochores using a single Cenp-A nucleosome (Cenp-ANuc), each of which is perfectly centred on its cognate centromere 7–9. The inner and outer kinetochore modules are responsible for interacting with centromeric chromatin and microtubules, respectively. Here, we describe the cryo-EM structure of the S. cerevisiae inner kinetochore module - the constitutive centromere associated network (CCAN) complex, assembled onto a Cenp-A nucleosome (CCAN–Cenp-ANuc). The structure explains the inter-dependency of CCAN’s constituent sub-complexes and shows how the ‘Y’-shaped opening of CCAN accommodates Cenp-ANuc to allow specific CCAN subunits to contact the nucleosomal DNA and histone subunits. Interactions with the unwrapped DNA duplex at the two termini of Cenp-ANuc are mediated predominantly by a DNA-binding groove present in the Cenp-LN sub-complex. Disruption of these interactions impairs assembly of CCAN onto Cenp-ANuc. Our data indicate a mechanism of Cenp-A nucleosome recognition by CCAN and how CCAN acts as a platform for assembly of the outer kinetochore for linking centromeres to the mitotic spindle for chromosome segregation.