Kinetochore Function and Cell Cycle Progression Revision

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DESCRIPTION (provided by applicant): Basic studies of genes required for the function of kinetochores and the surveillance of the spindle checkpoint are directly relevant to cancer research. Our goal is to identify and characterize proteins required for mitotic chromosome segregation in eukaryotes. The kinetochore, which consists of centromere DNA and associated proteins, is crucial for maintaining and segregating chromosomes during mitosis and meiosis. For these studies we will use the budding yeast Saccharomyces cerevisiae, as its process of mitotic division is comparable with that of multicellular eukaryotes. In Aim 1, we will characterize the functions of Dts proteins targeted by the spindle checkpoint. Our synthetic-lethality screen using a mad2-deletion mutant against a set of yeast deletion mutations identified 32 genes, including those encoding previously characterized kinetochore proteins, microtubule-binding proteins, chromatin-binding proteins, and cohesion proteins. We named the 4 previously uncharacterized genes DTS-1 through DTS-4. The dts3 mutants show a phenotype typical of kinetochore mutants, and Dts3 interacts with kinetochore proteins. We will characterize the function of Dts1, 2, and 4 by performing a series of genetic and biochemical assays. In Aim 2, we will investigate how Bub1 (a spindle checkpoint kinase) controls Sgo1 (Shugoshin, a tension sensor at the kinetochore). Sgo1 protects centromeric cohesin (Scc1/RAD21). BUB1 regulates the stability and centromeric localization of SGO1 in human cells. Although budding yeast Sgo1 does not regulate cohesion in mitosis, it is a tension sensor at kinetochores. It has recently been shown that the Bub1 kinase domain and Sgo1 act together to ensure the efficient bi-orientation of chromosomes; thus, both appear to be required for the tension checkpoint. However, the mechanism by which Bub1 controls Sgo1 function is unknown. We found that Bub1 interacts with Scc1 and phosphorylates Scc1. Thus, we hypothesize that Bub1 regulates Sgo1 function through Scc1 phosphorylation, and we will perform a series of experiments to test the hypothesis. Finally, in Aim 3 we will determine the molecular mechanism that regulates the spindle checkpoint during the cell cycle. The spindle checkpoint is deactivated and should not be activated during anaphase. Mutual inhibition between the anaphase-promoting complex (APC) and Mps1, an essential component of the spindle checkpoint, leads to sustained inactivation of the spindle checkpoint. However, how the APC is reactivated remains unclear. We have recently found that Bub1 is a target of the APC in yeast, and our preliminary data suggest that phosphorylated Bub1 is the preferred target of the APC during anaphase but not in G1. These results imply that the accumulation of phosphorylated Bub1 during metaphase is the signal that initiates the silencing of spindle checkpoint activity after a prolonged mitotic arrest. We will further characterize the mechanism of "adaptation" of the spindle checkpoint. PUBLIC HEALTH RELEVANCE: During cell division, chromosomes can be lost or gained when they do not segregate accurately. Having an abnormal number of chromosomes is called aneuploidy, and this situation can cause cancer to develop. Our studies on the mechanism of chromosome segregation will therefore contribute to the understanding of cancer development.