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© The Rockefeller University Press, 0021-9525/1997//1383 $5.00
The Journal of Cell Biology, Volume 139, Number 6, , 1997 1383-1396

Article

Probing the Architecture of a Simple Kinetochore Using DNA–Protein Crosslinking



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

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

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 p58CTF13 p64CEP3 and p110NDC10 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 p110NDC10 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.

Abbreviations used in this paper: BrdC, bromodeoxycytidine; BrdU, bromodeoxyuridine.

The work in this paper was supported by the National Institutes of Health Grant GM51464, NATO Grant CRG960763 and the Lucille P. Markey Charitable Trust. K.B. Kaplan is supported by the Cancer Research Fund of the Damon Runyon Walter Winchell Foundation Fellowship, DRG-1321.

Address all correspondence to Peter K. Sorger, Department of Biology, 68-371, MIT, 77 Massachusetts Avenue, Cambridge, MA 02139. Tel.: (617) 252-1648. Fax: (617) 253-8550. E-mail: psorger{at}mit.edu


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