Elucidating the mechanisms of kinetochore assembly using a novel assay

Thesis (Ph.D.)--University of Washington, 2018Cells rely upon the kinetochore in order to segregate their chromosomes during every cellular division. The kinetochore is a large, complex protein structure that must assemble onto the centromeric region of the chromosome and attach to the spindle microtubules. Therefore, it is imperative that cells accurately establish the centromere and build functional kinetochores in order to ensure proper segregation of the DNA. The centromeric nucleosome is marked by the conserved CENP-A histone. In order to restrict CENP-ACse4 incorporation to the centromere, the cellular levels of CENP-ACse4 in budding yeast are kept low by ubiquitination. I found that CENP-ACse4 is also post-translationally modified with SUMO, a small protein that is similar in many ways to ubiquitin. CENP-ACse4 is sumoylated by the Siz1 and Siz1 E3 SUMO ligases, and the sumoylation peaks during S phase of the cell cycle. I found that sumoylation does not effect CENP-ACse4 stability, but later work from another group suggested that sumoylation targets CENP-ACse4 for ubiquitination and subsequent degradation. It will be important to resolve these discrepancies and understand how CENP-ACse4 is regulated by this novel post-translational modification. In order to create a biochemical tool that could help address unanswered questions such as this, I developed an assay to assemble kinetochores in vitro onto a centromeric template. The cell-free assay I developed has the same fundamental requirements as kinetochore assembly in vivo, including a dependency upon the CENP-ACse4 chaperone, suggesting that a centromeric nucleosome forms onto the DNA template. Importantly, this de novo assembly method is sensitive to phosphoregulatory events and generates complete kinetochores that are capable of binding to microtubules. I used this assay to address why kinetochores recruit the key microtubule-binding Ndc80 complex through two pathways: the Mis12 complex and CENP-TCnn1. Although budding yeast CENP-TCnn1 is nonessential, I found that it becomes required for Ndc80 complex recruitment and cell viability when the Mis12 complex pathway to assembly is inhibited by phosphoregulation. Thus, CENP-TCnn1 can independently recruit the Ndc80 complex and is required to maintain intact kinetochores throughout the cell cycle. The potential applications for the kinetochore assembly assay are numerous and extend to many fundamental questions about centromere and kinetochore biology