The outer kinetochore comprises three subcomplexes, KNL1, MIS12, and NDC80, which are collectively referred to as the KMN network 9. CENP-C and another CCAN subunit, CENP-T, provide a platform for assembly of the outer layer of kinetochores 7, 8. The CENP-A nucleosome (CENP-A Nuc) is surrounded by a group of 16 proteins, the constitutive centromere-associated network (CCAN) and organized in various stable subcomplexes 5, 6, 7. However, the molecular details underlying kinetochore assembly and its functional coupling to chromosome movements remain less characterized. In fact, early electron microscopic (EM) analyses have indicated that the morphology of the outer kinetochore structure changes dynamically during mitotic progression 3, 4, suggesting that the kinetochore is dynamically assembled and functionally matured in mitosis. In eukaryotic cells, kinetochores, which are sophisticated molecular machines for cell division fate decisions, constitute a dynamic link between kinetochore microtubule attachment and spindle checkpoints, a signaling cascade that prevents chromosome segregation before completion of bi-orientation during cell division. Thus, these analyses provide new insights into mechanisms of action underlying kinetochore assembly and function in mitosis.Ĭorrect chromosome segregation relies on the centromere, a specialized chromatin domain present throughout the cell cycle that acts as a platform on which transient assembly of the kinetochore occurs during mitosis 1, 2. The DNA binding activity is essential for CENP-LN localization to centromere and chromosome segregation during mitosis. Further, CENP-N does not bind to the RG-loop of CENP-A but to DNA in the CCAN complex. The duplex DNA is topologically entrapped by human CCAN.

The overall structure relates to the cooperative interactions and interdependency of the constituent sub-complexes of the CCAN. Here, by use of cryo-electron microscopy and functional analyses, we reveal the molecular basis of how human CCAN interacts with duplex DNA and facilitates accurate chromosome segregation. However, the complex centromeres of human chromosomes comprise millions of DNA base pairs and attach to multiple microtubules. The centromeres of budding yeast, which are relatively simple, are connected to individual microtubules via a kinetochore constitutive centromere associated network (CCAN). In mitosis, accurate chromosome segregation depends on kinetochores that connect centromeric chromatin to spindle microtubules.

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