The accurate segregation of chromosomes at mitosis requires precise assembly and regulation of kinetochores, structures that form on centromeric DNA and attach chromosomes to the microtubules of the mitotic spindle. The long-term goals of this study are to determine the molecular architecture of the S. cerevisiae kinetochore, to identify and analyze the proteins that contribute to the assembly of microtubule attachment sites and to uncover the events that are monitored by the mitotic checkpoint. This will be accomplished by biophysical and genetic analysis of kinetochore proteins combined with in vitro reconstitution and characterization of centromere and microtubule binding activities. Specifically:Systematic genetic and biochemical analysis of the roughly 30 known kinetochore proteins will be undertaken, using efficient methods for manipulating genes and proteins. The regulation and assembly of the Dam1-Spc34-complex, an essential microtubule binding complex,, will be investigated, its components identified and its association with microtubules studied using biochemical methods. The structure and function of the Ndc80-complex, a core component of the kinetochore, will be analyzed and its role in the mitotic checkpoint established The molecular architecture of the CBF3 centromere-binding complex will be investigated and the reconstitution of CBF3-dependent microtubule attachment sites attempted in vitro. The significance of this work derives from its goal of generating a precise molecular model for kinetochore function. Kinetochores are poorly understood structures and the simplicity of the yeast kinetochore makes it particularly amenable to molecular analysis. Because many of the protein components and properties of kinetochores have been conserved through evolution, analysis of the relatively simple yeast kinetochore is expected to contribute significantly to our understanding of chromosome segregation in animal cells. Errors in chromosome segregation give rise to aneuploidy, a nearly universal property of tumor cells. Thus, the study of kinetochore biology has the potential to reveal how chromosome instability arises in human cancer.
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