Kinetochore specification and assembly requires the targeted deposition of specialized nucleosomes containing the histone H3 variant CENP-A at centromeres. However, CENP-A is not sufficient to drive full-kinetochore assembly, and it is not clear how centromeric chromatin is established. Here, we identify CENP-W as a component of the DNA-proximal constitutive centromere-associated network (CCAN) of proteins. We demonstrate that CENP-W forms a DNA-binding complex together with the CCAN component CENP-T. This complex directly associates with nucleosomal DNA and with canonical histone H3, but not with CENP-A, in centromeric regions. CENP-T/CENP-W functions upstream of other CCAN components with the exception of CENP-C, an additional putative DNA-binding protein. Our analysis indicates that CENP-T/CENP-W and CENP-C provide distinct pathways to connect the centromere with outer kinetochore assembly. In total, our results suggest that the CENP-T/CENP-W complex is directly involved in establishment of centromere chromatin structure coordinately with CENP-A.

KNL targets PP1 to kinetochores, where it antagonizes Aurora B activity.

Summary Mitotic chromosome segregation requires that kinetochores attach to microtubule polymers and harness microtubule dynamics to drive chromosome movement. In budding yeast, the Dam1 complex couples kinetochores with microtubule depolymerization. However, a metazoan homologue of the Dam1 complex has not been identified. To identify proteins that play a corresponding role at the vertebrate kinetochore-microtubule interface, we isolated a 3-subunit human Ska1 complex, including the previously uncharacterized protein Rama1, that localizes to the outer kinetochore and spindle microtubules. Depletion of Ska1 complex subunits severely compromises proper chromosome segregation. Reconstituted Ska1 complex possesses two separable biochemical activities; direct microtubule-binding through the Ska1 subunit, and microtubule-stimulated oligomerization imparted by the Rama1 subunit. The full Ska1 complex forms assemblies on microtubules that can facilitate the processive movement of microspheres along depolymerizing microtubules. In total, these results demonstrate a critical role for the Ska1 complex in interacting with dynamic microtubules at the outer kinetochore.