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. ? ?

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Scientist Development Award - Research & Training (K01)
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Subcommittee G - Education (NCI)
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Eckstein, David J
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University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
United States
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Varma, Dileep; Chandrasekaran, Srikripa; Sundin, Lynsie J R et al. (2012) Recruitment of the human Cdt1 replication licensing protein by the loop domain of Hec1 is required for stable kinetochore-microtubule attachment. Nat Cell Biol 14:593-603
Taylor, Sarah M; Nevis, Kathleen R; Park, Hannah L et al. (2010) Angiogenic factor signaling regulates centrosome duplication in endothelial cells of developing blood vessels. Blood 116:3108-17
Nevis, Kathleen R; Cordeiro-Stone, Marila; Cook, Jeanette Gowen (2009) Origin licensing and p53 status regulate Cdk2 activity during G(1). Cell Cycle 8:1952-63
Liu, Peijun; Slater, Damien M; Lenburg, Marc et al. (2009) Replication licensing promotes cyclin D1 expression and G1 progression in untransformed human cells. Cell Cycle 8:125-36
Dorn, Elizabeth S; Chastain 2nd, Paul D; Hall, Jonathan R et al. (2009) Analysis of re-replication from deregulated origin licensing by DNA fiber spreading. Nucleic Acids Res 37:60-9
Cook, Jeanette Gowen (2009) Replication licensing and the DNA damage checkpoint. Front Biosci (Landmark Ed) 14:5013-30
Sotillo, Elena; Garriga, Judit; Padgaonkar, Amol et al. (2009) Coordinated activation of the origin licensing factor CDC6 and CDK2 in resting human fibroblasts expressing SV40 small T antigen and cyclin E. J Biol Chem 284:14126-35
McCall, Chad M; Miliani de Marval, Paula L; Chastain 2nd, Paul D et al. (2008) Human immunodeficiency virus type 1 Vpr-binding protein VprBP, a WD40 protein associated with the DDB1-CUL4 E3 ubiquitin ligase, is essential for DNA replication and embryonic development. Mol Cell Biol 28:5621-33
Hall, Jonathan R; Lee, Hyun O; Bunker, Brandon D et al. (2008) Cdt1 and Cdc6 are destabilized by rereplication-induced DNA damage. J Biol Chem 283:25356-63
Hall, Jonathan R; Kow, Evelyn; Nevis, Kathleen R et al. (2007) Cdc6 stability is regulated by the Huwe1 ubiquitin ligase after DNA damage. Mol Biol Cell 18:3340-50

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