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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.
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