Abstract: Accurate chromosome segregation depends on the kinetochore, which is the complex of proteins that link microtubules to centromeric DNA. The kinetochore of the budding yeast Saccharomyces cerevisiae consists of more than 80 proteins assembled on a 125-bp region of DNA. 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 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, bi-orient sister kinetochores at metaphase during the mitotic cycle, segregate sister chromatids, and repair errors in chromosome attachment. We show the synthetic kinetochore 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 assembly of the complex structure that directs mitotic chromosome segregation.