Centromere-dependent binding of yeast minichromosomes to microtubules in vitro

Abstract

We present an in vitro assay for yeast centromere function; isolated yeast minichromosomes require a functional centromere to bind to bovine microtubules and sediment with them. Centromere-bovine microtubule complexes form at physiological microtubule concentrations. Two of the three centromere DNA elements, which are necessary for centromere function in vivo, are also necessary for centromeres to bind microtubules in vitro. However, purified centromere DNA alone does not bind to microtubules. These results suggest that microtubule binding must be mediated by the two centromere DNA elements and factors that associate with one or both of them. The percent of centromeres with microtubule-binding activity is 7- to 10-fold higher in lysates made from nocodazole-arrested G2-M cells than from alpha factor G1 cells, suggesting that this centromere activity is regulated during the cell cycle. The potential of this assay for dissecting centromere assembly, function, and regulation is discussed.