To maintain proper genome integrity, it is critical that cells properly coordinate intracellular and extracellular signaling pathways controlling DNA replication. Improper regulation of this fundamental process can ultimately contribute to inappropriate cell proliferation, apoptosis, and genome instability, which in turn promote the likelihood of tumorigenesis. Thus, a comprehensive understanding of the normal regulation of DNA replication as well as mechanisms contributing to its deregulation is critical in the development of novel therapies and diagnostic tools for cancer treatment. The research proposed here will focus on the very first regulatory step in eukaryotic DNA replication, which is the licensing of thousands of replication initiation sites, or origins, throughout the genome. Origins are rendered competent, or licensed, for DNA replication in S phase by the chromatin-loading of a DNA helicase known as the Mini-Chromosome Maintenance Complex (MCM) during G1 phase of the cell cycle. While MCM complexes are constitutively nuclear and can exist in both soluble and chromatin-bound states, only origins containing chromatin-bound MCM complexes are licensed for replication. This licensing step is tightly regulated such that it is ony allowed during G1 phase and prohibited during quiescence (also termed G0), S, G2, and M phases. Molecular mechanisms coordinating these changes in MCM loading status with cell cycle progression remain unclear and are the focus of this proposal.
The first aim seeks to determine how unlicensed chromatin is maintained during cellular quiescence. Our lab recently discovered that the stress MAP kinases p38 and JNK inhibit licensing during a cellular stress response, and these kinases are highly active in normal cells during quiescence. Based on these observations, we hypothesize that the stress MAPKs additionally play a role in blocking MCM loading during quiescence to maintain the non-proliferative state. We will test this idea by inhibiting MAPK activity during quiescence and observing effects on licensing competence.
The second aim of this proposal addresses the question of how MCM loading in G1 is linked with S phase entry. Given our recent finding that MCM loading is required for initiation of S phase, we postulate that as-yet unidentified proteins interact preferentially with loaded MCM complexes and function to promote the G1/S transition. To discover such mediators of the G1/S transition, we will isolate MCM complexes from soluble and chromatin fractions of G1 cells for comparative mass spectrometry analysis. Proteins interacting exclusively with chromatin-bound MCM complexes in G1 will be the focus of further functional analyses to determine their role in mediating cell cycle progression. Ultimately, we anticipate that the long-term analysis of newly identified origin licensing regulators from these studies will guide the development of novel therapeutics for many human cancers.

Public Health Relevance

Human cancers develop as a consequence of uncontrolled cell cycle entry and DNA replication. While normal cells have mechanisms in place to avoid inappropriate DNA replication, cancer cells lack these mechanisms and proliferate inappropriately. To better understand how these regulatory processes work, my project will identify previously undiscovered protein factors that coordinate the initiation of DNA replication with proliferative and anti-proliferative cues. Ultimately, our goal is to understand what contributes to the deregulation of cell proliferation in cancer cells and how this information coul be used to guide the development of novel anticancer therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
1F31CA165891-01A1
Application #
8457662
Study Section
Special Emphasis Panel (ZRG1-F05-R (20))
Program Officer
Korczak, Jeannette F
Project Start
2013-04-01
Project End
2016-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
1
Fiscal Year
2013
Total Cost
$29,692
Indirect Cost
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