Factors required for the binding of reassembled XXXX kinetochores to microtubules in vitro.

@entity67444 @entity375 @entity18682
Kinetochores are structures that assemble on centromeric DNA and mediate the attachment of chromosomes to the microtubules of the mitotic spindle. The protein components of kinetochores are poorly understood, but the simplicity of the @entity375 kinetochore makes it an attractive candidate for molecular dissection. Mutations in genes encoding @entity18682 and @entity67444 , proteins that bind to @entity375 centromeres, interfere with chromosome segregation in vivo. To determine the roles played by these factors and by various regions of centromeric DNA in kinetochore function, we have developed a method to partially reassemble kinetochores on exogenous centromeric templates in vitro and to visualize the attachment of these reassembled kinetochore complexes to microtubules. In this assay, single reassembled complexes appear to mediate microtubule binding. We find that @entity67444 is absolutely essential for this attachment but, contrary to previous reports (Hyman, A. A., K. Middleton, M. Centola, T.J. Mitchison, and J. Carbon. 1992. Microtubule-motor activity of a @entity375 centromere-binding protein complex. Nature (Lond.). 359:533-536) is not sufficient. Additional cellular factors interact with @entity67444 to form active microtubule-binding complexes. This is mediated primarily by the CDEIII region of centromeric DNA but CDEII plays an essential modulatory role. Thus, the attachment of kinetochores to microtubules appears to involve a hierarchy of interactions by factors that assemble on a core complex consisting of DNA-bound @entity67444 .
