Native yeast kinetochore structures identify an essential inner kinetochore interaction

Abstract

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 developed a one-step protocol to purify native inner kinetochore subcomplexes for structural studies. We performed cryo-electron microscopy on the purifications and generated density maps of four separate inner kinetochore complexes, two of which had not previously been visualized and may represent intermediate assembly states. We identified 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.