PHOSPHOREGULATION OF DNA REPLICATION ORIGIN LICENSING IN MAMMALIAN CELLS This proposal seeks new insight into the fundamental organization of the human cell division cycle and how perturbations to that organization lead to genome instability and pathological states. Complete and efficient duplication of the entire human genome requires that many thousands of DNA replication origins become ?licensed? in G1 of each cell division cycle through the loading of MCM helicase complexes. DNA-loaded MCM complexes are then activated during S phase. Loss of normal origin licensing control causes hypersensitivity to replication stress and induces genome instability, which can ultimately lead to oncogenesis, developmental defects, and degeneration. Our long-term goal is to understand how DNA replication origin licensing control is coordinated with intracellular and extracellular signaling pathways that control proliferation and development. We hypothesize that perturbations to this coordination cause genome instability and proliferation failure. Our experimental approach is a combination of quantitative single cell analyses with molecular biology and biochemistry using cultured human cells. We focus on uncovering molecular mechanisms and dynamics, and then testing the cellular consequences of disrupting those mechanisms. Our recent progress, innovative experimental strategies, and preliminary data inspire a new series of projects to address these specific goals: 1) determine precisely how changes in individual cyclin/CDK enzymes impact origin licensing and the G1 to S phase transition 2) define the molecular consequences phosphorylating an essential origin licensing protein, Cdt1, and 3) define the relationships among CDK-mediated phosphorylation, the APC/C E3 ubiquitin ligase, and origin licensing, particularly for regulation of the Cdc6 licensing protein and preventing re-replication. Success will shed light on the mechanisms that can drive mutagenesis, cancer, cell death, and aging. The deep understanding of cell proliferation control sought through the pursuit of these aims will have downstream implications for efforts to precisely define and treat human disease and for future regenerative therapies.

Public Health Relevance

One crucial step in the cell division cycle is the complete and precise replication of chromosomal DNA, and errors in DNA replication control can cause developmental abnormalities, cell death, or cancer. We will address pressing unanswered questions about how the earliest irreversible step in DNA replication is regulated by cellular master control enzymes. These control enzymes are frequently deregulated in diseases such as cancer, so understanding the exact consequences of their action will help design future precision diagnostics and therapies to treat cancer and degenerative diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Reddy, Michael K
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University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
United States
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Matson, Jacob P; Cook, Jeanette G (2017) Cell cycle proliferation decisions: the impact of single cell analyses. FEBS J 284:362-375
Chao, Hui Xiao; Poovey, Cere E; Privette, Ashley A et al. (2017) Orchestration of DNA Damage Checkpoint Dynamics across the Human Cell Cycle. Cell Syst 5:445-459.e5
Grant, Gavin D; Cook, Jeanette G (2017) The Temporal Regulation of S Phase Proteins During G1. Adv Exp Med Biol 1042:335-369
Matson, Jacob Peter; Dumitru, Raluca; Coryell, Philip et al. (2017) Rapid DNA replication origin licensing protects stem cell pluripotency. Elife 6:
Mulvaney, Kathleen M; Matson, Jacob P; Siesser, Priscila F et al. (2016) Identification and Characterization of MCM3 as a Kelch-like ECH-associated Protein 1 (KEAP1) Substrate. J Biol Chem 291:23719-23733
Pozo, Pedro N; Cook, Jeanette Gowen (2016) Regulation and Function of Cdt1; A Key Factor in Cell Proliferation and Genome Stability. Genes (Basel) 8:
Coleman, Kate E; Grant, Gavin D; Haggerty, Rachel A et al. (2015) Sequential replication-coupled destruction at G1/S ensures genome stability. Genes Dev 29:1734-46
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