Precise duplication of the entire human genome requires that thousands of origins are licensed and then initiate replication exactly once per cell cycle. Origin licensing is accomplished through the assembly of prereplication complexes at origins during the G1 phase of the cell cycle. Licensing too few origins results in incomplete replication, and licensing origins that have already been replicated results in rereplication. Both situations are sources of genome instability that can promote aberrant cell proliferation and oncogenesis. Replication initiation is stimulated by the activity of cyclin dependent protein kinases (Cdks) that are activated at the G1/S phase transition and remain active until mitosis. To prevent any sequence from being replicated more than once, origin licensing is inhibited once replication initiation begins in S phase. The mechanisms that inhibit origin licensing after G1 to avoid rereplication include a critical contribution from the same cyclin dependent protein kinases (Cdks) that stimulate replication initiation. These Cdks function to block the assembly of prereplication complexes after G1. A consequence of the dual function of Cdks in replication control is that origin licensing must be fully completed in G1 (during the period of low Cdk activity) before S phase begins so that the entire genome can be replicated, but it is not known how cells ensure that origin licensing is complete before S phase begins. A second consequence of the role of Cdks in preventing inappropriate re-licensing of origins is that many environmental changes trigger cell cycle checkpoints that arrest the cell cycle by inhibiting Cdks. If such changes - such as DNA damage or cellular stress - are encountered after S phase begins, then cells risk rereplication once they recover from the checkpoint arrest. To avoid rereplication and its associated genome instability, such cell cycle checkpoints must also inhibit origin licensing by mechanisms that are independent of Cdk activity. This proposal addresses three distinct interfaces between replication licensing control and individual checkpoint responses.
The aims are to 1) Determine the mechanism that links origin licensing to Cdk activation and S phase entry, 2) Determine the mechanism controlling release of the licensing protein, Cdc6, from origins after DNA damage, and 3) Determine the mechanism that prevents origin licensing during a cellular stress response. Our methods rely on the manipulation of specific proteins and activities in cultured human cells. We evaluate the effects of these manipulations on the abundance, activity and localization of essential replication and checkpoint proteins, and we determine the effects of these manipulations on protein- protein and protein-DNA interactions. We anticipate that a comprehensive understanding of the regulation of origin licensing will facilitate the diagnosis, classification, and treatment of human cancers as well as other diseases that involve aberrant cell proliferation.

Public Health Relevance

Although it is clear that cancer cells proliferate inappropriately, it is still not fully understood how these cells differ from normal cells or what controls their progression to metastatic disease. Errors in the regulation of DNA replication are major contributors to cancer development, and this proposal is designed to gain new insight into the regulation of the first step in DNA replication, the localization of essential replication proteins to DNA. The specific focus is on the coordination of this initial step with the cellular responses to perturbations in the intracellular or extracellular environment.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM083024-04
Application #
8204999
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Reddy, Michael K
Project Start
2009-01-01
Project End
2013-12-31
Budget Start
2012-01-01
Budget End
2012-12-31
Support Year
4
Fiscal Year
2012
Total Cost
$264,562
Indirect Cost
$83,243
Name
University of North Carolina Chapel Hill
Department
Biochemistry
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Lane, Karen R; Yu, Yanbao; Lackey, Patrick E et al. (2013) Cell cycle-regulated protein abundance changes in synchronously proliferating HeLa cells include regulation of pre-mRNA splicing proteins. PLoS One 8:e58456
Dorn, Elizabeth Suzanne; Cook, Jeanette Gowen (2011) Nucleosomes in the neighborhood: new roles for chromatin modifications in replication origin control. Epigenetics 6:552-9
Chandrasekaran, Srikripa; Tan, Ting Xu; Hall, Jonathan R et al. (2011) Stress-stimulated mitogen-activated protein kinases control the stability and activity of the Cdt1 DNA replication licensing factor. Mol Cell Biol 31:4405-16
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