Biochemical and molecular genetic approaches will be used in budding yeast to explore the mechanisms that control progression through mitosis, the final phase of the cell cycle. The separation of the duplicated chromosomes in mitosis and the subsequent events of mitotic exit are regulated by a large multisubunit enzyme called the anaphase-promoting complex or APC. The APC is a ubiquitin-protein ligase that catalyzes the final step in the ubiquitination of several key mitotic regulatory proteins, resulting in their proteolytic destruction. The APC is capable of processive ubiquitination - that is, it can modify its targets multiple times during a single enzyme-substrate interaction. Our recent work indicates that the APC subunit Docl is required for APC processivity. We hypothesize that Doc1 promotes processivity by enhancing substrate binding to the APC, perhaps by directly interacting with the substrate. Experiments proposed in the first aim of this proposal will test this hypothesis. The affinity of APC-substrate interactions will be measured in the presence and absence of Doc1, and a putative ligand-binding domain in Doc1 will be mutagenized to explore its function in substrate binding. Crosslinking methods will be used to assess interactions between the substrate and Doc1 and also to identify APC subunits that interact with Cdh1, another APC subunit known to be involved in substrate binding. The structure of the APC will also be explored in meiosis. Studies in the second aim will address the regulation and function of the polo-related protein kinase Cdc5, another important regulator of late mitotic events and the APC. Proteins that associate with Cdc5 or regulate its activity in the cell will be identified. In total, the proposed experiments will lead to a more complete knowledge of the mechanisms that control chromosome segregation and mitotic exit in eukaryotes. Such knowledge is an essential prerequisite for an understanding of the segregation errors that often contribute to developmental defects or cancer progression.
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