Native yeast kinetochore structures identify an essential inner kinetochore interaction

Kinetochores must accurately assemble on centromeres for faithful chromosome segregation. Although a conserved centromeric nucleosome is essential for kinetochore assembly, budding yeast centromeric DNA is a poor template for nucleosome formation in vitro, perhaps due to its intrinsic rigidity. To better understand yeast inner kinetochore assembly, we develop a one-step protocol to purify native inner kinetochore subcomplexes for structural studies. We perform cryoelectron microscopy on the purifications and generate density maps of four separate inner kinetochore complexes, two of which have not been previously visualized and may represent intermediate assemblage states. We identify an Ndc10 trimerization domain that engages centromeric DNA and a pair of CBF3 complexes and is associated with substantial bending of centromeric DNA. Ndc10 trimerization is essential for kinetochore assembly and chromosome segregation. We propose that Ndc10 trimerization facilitates centromeric DNA bending to stabilize the centromeric nucleosome and inner kinetochore. Accurate kinetochore assembly at centromeres is essential for proper chromosome segregation. Here, the authors purified and visualized native yeast inner kinetochore complexes, revealing that Ndc10 trimerization bends centromeric DNA and is required to stabilize kinetochore assembly and chromosome segregation. We are grateful to the Biggins and Asbury labs for critical reading of the manuscript and fruitful discussions. We thank Chip Asbury and Barry Stoddard for insightful comments on the manuscript. We thank the Proteomics & Metabolics shared resource fa... [2151 chars]