Budding yeast centromere composition and assembly as revealed by in vivo cross-linking

Pamela B. Meluh, Douglas Koshland

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3401-3412
Number of pages12
JournalGenes and Development
Volume11
Issue number24
DOIs
StatePublished - Dec 15 1997

Keywords

  • CENP-C
  • CSE4
  • Centromere
  • Chromatin immunoprecipitation
  • Kinetochore
  • MIF2

ASJC Scopus subject areas

  • Genetics
  • Developmental Biology

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