The initiation of DNA replication is a crucial event required for cell proliferation in both normal cells and cancer cells. Many of the proteins that are central players at replication origins are excellent diagnostic markers for proliferation and potential targets for anti-proliferation therapies. We propose to investigate the function and regulation of two of these factors, Cdc6 and Cdt1, in intact mammalian cells. Cdc6 and Cdt1 are required for the formation of pre-replication complexes through recruitment of the Mcm complex to chromatin, and both Cdc6 and Cdt1 are subject to cell cycle control of expression and activity. Cdt1 protein is negatively regulated by the protein geminin, whereas the Cdc6 protein is regulated in part by cyclin dependent kinases. We have developed an experimental approach using recombinant adenoviral transduction that allows expression of normal or mutationally altered forms of these proteins in quiescent mammalian cells where replication complexes are absent. Using this system we have shown that Cdc6 expression is sufficient to induce the formation of functional pre-replication complexes and to stimulate DNA replication in cooperation with cyclin E/cdk2. Furthermore, we have demonstrated that geminin interferes with pre-replication complex formation, and that increased expression of Cdt1 can overcome this block. We will use these reagents and the functional assays we have developed to explore the molecular events that take place during replication initiation in mammalian cells. In particular, we propose experiments to accomplish the following goals: 1) Delineate the specific functional motifs in Cdc6 with particular emphasis on protein interactions, recruitment of Mcm proteins to chromatin, and Cdk-mediated phosphorylation; 2) Identify proteins that associate with Cdc6 and determine their functions; and 3) Determine the roles of Cdt1 using functional assays and focused two-hybrid strategies. I will initiate these studies under the mentorship of Professor Joseph R. Nevins and transition to an independent position during the second year of the award. My long-term goals are to direct an independent laboratory devoted to studying the regulation of DNA replication in which we take advantage of the powerful molecular genetics available in yeast alongside informative cell biological and biochemical functional studies in mammalian cells. ? ?
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