Probing the architecture of a simple kinetochore using DNA-protein crosslinking

Christopher W. Espelin, Kenneth B. Kaplan, Peter K. Sorger

Research output: Contribution to journalArticle

68 Scopus citations

Abstract

In budding yeast, accurate chromosome segregation requires that one and only one kinetochore assemble per chromosome. In this paper, we report the use of DNA-protein crosslinking and nondenaturing gel analysis to study the structure of CBF3, a four-protein complex that binds to the essential CDEIII region of Saccharomyces cerevisiae centromeres. We find that three subunits of CBF3 are in direct contact with CDEIII over a region of DNA that spans 80 bp. A highly asymmetric core complex containing p58(CTF13) p64(CEP3) and p110(NDC10) in direct contact with DNA forms at the genetically defined center of CDEIII. This core complex spans ~56 bp of CEN3. An extended complex comprising the core complex and additional DNA-bound p110(NDC10) also forms. It spans ~80 bp of DNA. CBF3 makes sequence-specific and - nonspecific contacts with DNA. Both contribute significantly to the energy of CBF3-DNA interaction. Moreover, important sequence-specific contacts are made with bases that are not conserved among yeast centromeres. These findings provide a foundation for understanding the organization of the CBF3- centromere complex, a structure that appears to initiate the formation of microtubule attachment sites at yeast kinetochores. These results also have implications for understanding centromere-binding proteins in higher cells.

Original languageEnglish (US)
Pages (from-to)1383-1396
Number of pages14
JournalJournal of Cell Biology
Volume139
Issue number6
DOIs
StatePublished - Dec 15 1997
Externally publishedYes

ASJC Scopus subject areas

  • Cell Biology

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