PROBING THE ARCHITECTURE OF A SIMPLE KINETOCHORE USING DNA-PROTEIN CROSS-LINKING

In budding yeast, accurate chromosome segregation requires that one an d only one kinetochore assemble per chromosome, In this paper, we repo rt the use of DNA-protein crosslinking and nondenaturing gel analysis to study the structure of CBF3, a four-protein complex that binds to t he essential CDEIII region of Saccharomyces cerevisiae centromeres, We find that three subunits of CBF3 are in direct contact with CDEIII ov er a region of DNA that spans 80 bp. A highly asymmetric core complex containing p58(CTF13) p64(CEP3) and p110(NDC10) in, direct contact wit h DNA forms at the genetically defined center of CDEIII. This core com plex spans similar to 56 bp of CEN3. An extended complex comprising th e core complex and additional DNA-bound p110(NDC10) also forms. It spa ns similar to 80 bp of DNA. CBF3 makes sequence-specific and -nonspeci fic contacts with DNA. Both contribute significantly to the energy of CBF3-DNA interaction, Moreover, important sequence-specific contacts a re made with bases that are not conserved among yeast centromeres. The se 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. Thes e results also have implications for understanding centromere-binding proteins in higher cells.