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

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

Research output: Contribution to journalArticlepeer-review

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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