CEP3 ENCODES A CENTROMERE PROTEIN OF SACCHAROMYCES-CEREVISIAE

We have designed a screen to identify mutants specifically affecting k inetochore function in the yeast Saccharomyces cerevisiae. The selecti on procedure was based on the generation of ''synthetic acentric'' min ichromosomes. ''Synthetic acentric'' minichromosomes contain a centrom ere locus, but lack centromere activity due to combination of mutation s in centromere DNA and in a chromosomal gene (CEP) encoding a putativ e centromere protein. Ten conditional lethal cep mutants were isolated , seven were found to be alleles of NDC10 (CEP2) encoding the 110-kD p rotein of yeast kinetochore. Three mutants defined a novel essential g ene CEP3. The CEP3 product (Cep3p) is a 71-kD protein with a potential DNA-binding domain (binuclear Zn-cluster). At nonpermissive temperatu re the cep3 cells arrest with an undivided nucleus and a short mitotic spindle. At permissive temperature the cep3 cells are unable to suppo rt segregation of minichromosomes with mutations in the central part o f element III of yeast centromere DNA. These minichromosomes, when iso lated from cep3 cultures, fail to bind bovine microtubules in vitro. T he sum of genetic, cytological and biochemical data lead us to suggest that the Cep3 protein is a DNA-binding component of yeast centromere. Molecular mass and sequence comparison confirm that Cep3p is the p64 component of centromere DNA binding complex Cbf3 (Lechner, 1994).