Kinetochore Function and Cell Cycle Progression

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DESCRIPTION (provided by applicant): Aneuploidy occurs in cancer cells when errors in chromosome segregation happen. Mutations leading to increased chromosome missegregation in certain cancers might be predisposing factors that accelerate tumorigenesis. By this model, the kinetochore and its regulatory system, which are essential for genome stability, are crucial in protecting against cancer development. The project's goal is to identify and characterize proteins required for mitotic chromosome segregation in eukaryotes, by using Saccharomyces cerevisiae as an experimental organism. Sgt1 and the core kinetochore protein, Skp1, promote assembly of the kinetochore core complex (CBF3) by activating Ctf13. Moreover, Skp1 and Sgt1 are part of the SCF complex (an E3 ubiquitin ligase). Specific Aim 1 is to investigate the biological function of Sgt1. This will be investigated by determining the time at which Sgt1 binds to CEN, and analyzing Sgt1's phosphorylation status. To test the hypothesis that Sgt1 and Skp1 are key components in the potential connection between kinetochore activation and ubiquitin-mediated degradation via the SCF complex, extragenic suppressor screens will be performed. Specific Aim 2 is to isolate and characterize proteins that interact with Sgt1. Immunoprecipitation mass spectrometry will be performed to isolate Sgt1 interactors. The function(s) of the isolated proteins will be characterized in genetics and biochemistry using the mutant strains that we will construct. Signaling from defective kinetochores to the mitotic checkpoint is thought to occur but is poorly understood. The principal investigator has shown that the spindle checkpoint protein Bub1 binds to Skp1 and that Bub1 associates with CEN DNA via Skp1. Therefore, Specific Aim 3 is to characterize the molecular interaction between kinetochores and the mitotic spindle checkpoint, especially the Bub1-Skp1 interaction. The contribution of the Bub1-Skp1 complex to the G2/M delays caused by mitotic defects, Bub1's Skp1 binding and CEN-associating domains, and additional Bub1 interactors will be analyzed. This work will reveal kinetochore functions of Skp1 and Sgt1 and novel kinetochore or cell-cycle regulators that can serve as entry points for further analysis of kinetochores in chromosome transmission and cell-cycle progression Achieving these aims will further elucidate mechanisms of cancer development.