Budding yeast centromere composition and assembly as revealed by in vivo cross-linking.
The centromere-kinetochore complex is a specialized chromatin structure that mediates bipolar attachment of replicated chromosomes to the mitotic spindle, thereby ensuring proper sister chromatid separation during 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 protein of the budding yeast Saccharomyces cerevisiae, previously implicated in centromere function by genetic criteria, resides specifically at centromeric loci in vivo. This provides definitive evidence for structural conservation between yeast and mammalian centromeres, as Mif2p 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 centromeric DNA in vivo. By examining Mif2p, Ndc10p, and Cbf1p association with centromeric DNA derivatives, we demonstrate the existence of centromeric subcomplexes that may correspond to assembly intermediates. Based on these observations, we provide a simple model for centromere assembly. Finally, given the sensitivity of this technique, its application to other sequence-specific protein-DNA complexes within the cell, such as origins of replication and enhancer-promoter regions, could be of significant value.
Meluh
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Howard Hughes Medical Institute, Carnegie Institution of Washington, Department of Embryology, Baltimore, Maryland 21210, USA. meluh@mail1.ciwemb.edu
Koshland
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D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002503:Centromere / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004271:DNA, Fungal / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005656:Fungal Proteins / Q000378:metabolism*; D018386:Kinetochores; D009687:Nuclear Proteins / Q000378:metabolism; D011233:Precipitin Tests; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C064042:CBF1 protein, S cerevisiae; C081071:CBF2 protein, S cerevisiae; D003432:Cross-Linking Reagents; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C083768:MIF2 protein, S cerevisiae; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins