A complete understanding of the complexities of cellular differentiation and neoplasia will require a thorough analysis of the molecular events occurring during eukaryotic cell division. One important aspect of the cell division process, namely, the accurate segregation of the replicated chromosomes to parent and daughter cells, is the subject of this proposal. The long range objectives of our research are to define the structural organization of the centromere regions of eukaryotic chromosomes, and to understand the molecular mechanisms operative in chromosome segregation in mitosis and meiosis. The research supported by this grant deals with the molecular mechanics and regulation of centromere function in the budding yeast Saccharomyces cerevisiae. The functional centromere DNA (CEN) in yeast is comparatively simple (about 125 bp) and thus is an excellent experimental model to analyze the mechanism of kinetochore function.
Our specific aims are to isolate and characterize all protein components of the yeast centromere/kinetochore,and to elucidate their functional roles in mediating microtubule attachment, sister chromatid attachment and release, and chromosome movement during cell division. For CEN DNA-binding proteins (CBF1, CBF3A, CBF3B, CBF3C) are now known. The genes specifying the three subunits of CBF3, an important multisubunit protein complex that binds specifically to the yeast centromere DNA, will be isolated and characterized. Combined genetic and molecular strategies will be used to define the mechanism of action of CBF3 and identify other protein factors involved in yeast centromere function. The comparative structures of various in vitro-assembled CEN DNA-protein complexes will be examined by nuclease protection experiments and by visualization in the electron micro- centromere from the purified components in vitro. We will attempt to obtain in vitro attachment of microtubules to CEN DNA- protein complexes assembled from purified components. The CBF1-CBF3 - CEN DNA complex will be used as a substrate to identify in fractionated yeast extracts other factors necessary to bring about microtubule attachment. Microtubule-dependent motor activity of yeast centromere proteins and DNA- protein complexes will be assayed. The role of protein kinases, phosphatases, or other regulatory factors in controlling the activity of the centromere proteins throughout the cell cycle will be assessed.
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