The accurate segregation of chromosomes requires precise control over the assembly of kinetochores, the structures that anchor and move chromosomes along the microtubules of the mitotic spindle. The goal of this work is to identify the proteins that comprise the Saccharomyces cerevsisiae kinetochore and to determine how they assemble on centromeric DNA and bind to microtubules. This is accomplished by reassembling kinetochore complexes in vitro with recombinant proteins and proteins purified from yeast extracts and then measuring centromere and microtubule binding activities. The biochemical properties of the reassembled kinetochores are determined and, using yeast genetics, these properties are related to the functions of kinetochores in vivo. In the long term, it is hoped that kinetochore microtubule attachment sites similar to those found in living cells can be reconstituted in vitro from fully purified components. The binding of the four-protein CBF3 complex to centromeric DNA appears to be the initiating event in kinetochore assembly. CBF3 proteins (p23Skp1, p58Ctf13, p64Cep3 and p110Ndc10) are tightly regulated by phosphorylation and by ubiquitin-dependent-degradation. To investigate this regulation and to identify CBF3-associated microtubule binding proteins, the following specific experiments will be performed: (1) The binding of CBF3 to wild-type and mutant centromere templates will be analyzed in vivo and in vitro. A specific model for the structure of the CBF3-centromere complex will be tested, as will the hypothesis that the functional CBF3 binding site is substantially larger than has been suggested by genetic studies to date. (2) The domains of CBF3 proteins involved in CBF3 assembly and in binding to DNA will be determined. (3) The regulation of CBF3 will be examined and the hypothesis that p58 is regulated by a coupled cycle of p23Skp1 dependent phosphorylation and ubiquitin-dependent degradation will be tested. (4) CBF3-associated proteins involved in the formation of a microtubule attachment site will be isolated and analyzed in vitro and in vivo. The hypothesis that the centromere-binding protein Mif2p binds to CBF3 will be tested biochemically. The analysis of structures such as kinetchores, involved in ensuring the fidelity of chromosome segregation, will be essential for understanding how aneuploidy and genetic instability arise in tumour cells.
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