CENTROMERE FUNCTION ON MINICHROMOSOMES ISOLATED FROM BUDDING YEAST

Centromeres are a complex of centromere DNA (CEN DNA) and specific fac tors that help mediate microtubule-dependent movement of chromosomes d uring mitosis. Minichromosomes can be isolated from budding yeast in a way that their centromeres retain the ability to bind microtubules in vitro. Here, we use the binding of these minichromosomes to microtubu les to gain insight into the properties of centromeres assembled in vi vo. Our results suggest that neither chromosomal DNA topology nor prox imity of telomeres influence the cell's ability to assemble centromere s with microtubule-binding activity. The microtubule-binding activity of the minichromosome's centromere is stable in the presence of compet itor CEN DNA, suggesting that the complex between the minichromosome C EN DNA and proteins directly bound to it is very stable. The efficienc y of centromere binding to microtubules is dependent upon the concentr ation of microtubule polymer and is inhibited by ATP. These properties are similar to those exhibited by mechanochemical motors. The binding of minichromosomes to microtubules can be inactivated by the presence of 0.2 M NaCl and then reactivated by restoring NaCl to 0.1 M. In 0.2 M NaCl, some centromere factor(s) bind to microtubules, whereas other (s) apparently remain bound to the minichromosome's CEN DNA. Therefore , the yeast centromere appears to consist of two domains: the first co nsists of a stable core containing CEN DNA and CEN DNA-binding protein s; the second contains a microtubule-binding component(s). The molecul ar functions of this second domain are discussed.