BUDDING YEAST CENTROMERE COMPOSITION AND ASSEMBLY AS REVEALED BY IN-VIVO CROSS-LINKING

The centromere-kinetochore complex is a specialized chromatin structur e that mediates bipolar attachment of replicated chromosomes to the mi totic spindle, thereby ensuring proper sister chromatid separation dur ing anaphase. The manner in which this important multimeric structure is specified and assembled within chromatin is unknown. Using in vivo cross-linking followed by immunoprecipitation, we show that the Mif2 p rotein of the budding yeast Saccharomyces cerevisiae, previously impli cated in centromere function by genetic criteria, resides specifically at centromeric loci in vivo. This provides definitive evidence for st ructural conservation between yeast and mammalian centromeres, as Mif2 p shares homology with CENP-C, a mammalian centromere protein. Ndc10p and Cbf1p, previously implicated in centromere function by genetic and in vitro biochemical assays, were also found to interact with centrom eric DNA in vivo. By examining Mif2p, Ndc10p, and Cbf1p association wi th centromeric DNA derivatives, we demonstrate the existence of centro meric subcomplexes that may correspond to assembly intermediates. Base d on these observations, we provide a simple model for centromere asse mbly. finally, given the sensitivity of this technique, its applicatio n to other sequence-specific protein-DNA complexes within the cell, su ch as origins of replication and enhancer-promoter regions, could be o f significant value.