PROJECT SUMMARY/ABSTRACT The proposed research explores the fascinating regulatory system that governs the eukaryotic cell division cycle. The cell-cycle control system is based on master regulatory enzymes that include the cyclin-dependent protein kinases (Cdks) and a ubiquitin ligase called the anaphase-promoting complex or cyclosome (APC/C). As the cell progresses through the steps of cell division, sequential Cdk-cyclin complexes modify hundreds of proteins, leading to chromosome duplication in S phase and alignment of duplicated chromosome on the mitotic spindle in M phase. The APC/C then triggers destruction of several key regulators, unleashing the dramatic events of chromosome segregation and cytokinesis. One critical APC/C substrate is securin, an inhibitor of the protease, separase; APC/C-mediated securin destruction releases separase to cleave the cohesin complex holding duplicated chromosomes together. The precise timing of cell cycle events requires that Cdks and the APC/C are activated and modify their targets in a specific order. The biochemical mechanisms underlying this order are the central focus of the research described in this application. The work will explore a diverse array of interesting mechanistic problems in Cdk and APC/C function. Studies in budding yeast will address the complex information processing that can be achieved through multi-site phosphorylation of Cdk substrates, focusing on transcriptional regulators that govern cyclin gene expression. Multiple lines of investigation will address the mechanisms by which the APC/C, together with its substrate-binding activator subunit, controls the destruction of specific targets. A newly developed single-molecule approach will provide rigorous quantitative measurements of APC/C-substrate interactions, providing insight into the variations in substrate affinity that underlie the ordering of cell cycle events. Biochemical and structural approaches with human proteins will be used to unravel the mechanisms by which the APC/C activator subunit is loaded onto the APC/C by the chaperonin TRiC/CCT. Similar approaches will be used to determine the mechanisms by which human separase recognizes its substrates, and the mechanisms by which securin and other regulatory proteins inhibit separase catalytic function. The information gained from these studies will provide important new insights into the control of cell-cycle progression, and will thereby enhance our understanding of diseases, such as cancer, in which cell-cycle control or chromosome segregation is defective. These studies will also illuminate general regulatory mechanisms of major importance throughout cell biology and human disease.