Accurate chromosome segregation depends on the kinetochore, the complex of proteins that link microtubules to centromeric DNA1. The budding yeast kinetochore consists of more than 80 proteins assembled on a 125bp region of DNA1. We studied the assembly and function of kinetochore components by fusing individual kinetochore proteins to the lactose repressor (LacI) and testing their ability to improve the segregation of a plasmid carrying tandem repeats of the lactose operator (LacO). Targeting Ask1, a member of the Dam1/DASH microtubule-binding complex, creates a synthetic kinetochore that performs many functions of a natural kinetochore: it can replace an endogenous kinetochore on a chromosome, biorient sister kinetochores at metaphase of mitosis, segregate sister chromatids, and repair errors in chromosome attachment. We show the synthetic kinetochore’s functions do not depend on the DNA-binding components of the natural kinetochore but do require other kinetochore proteins. We conclude that tethering a single kinetochore protein to DNA triggers the assembly of the complex structure that directs mitotic chromosome segregation.
