The overall goal of this project is to understand the mechanism of DNA replication through difficult-to-replicate sites of DNA. These would include sites predicted to form DNA secondary structure such as telomeres and fragile sites. Telomeres consist of repetitive, G-C rich DNA and have long been considered natural impediments to the replication fork machinery. Fragile sites are expressed as gaps or breaks in chromosomal DNA that occur during conditions of replication stress. Telomeres and fragile sites are known to cause genomic instability and can lead to carcinogenesis when not properly maintained. CTC1-STN1-TEN1 (CST) is a novel protein complex, which was recently discovered in plants and vertebrates. STN1 and TEN1 are homologues to yeast proteins, which are involved in telomere maintenance and end-protection. In humans, CST was shown to localize to telomeres. Depletion of human CST subunits results in hallmarks of genomic instability, i.e. increased 3H2AX foci and chromatin bridges, as well as telomere abnormalities such as increased G-overhangs and telomere signal loss. Results from several labs suggest that CST has both telomeric and non- telomeric functions. Importantly, CST was shown to interact with DNA polymerase 1-primase (pol 1), which initiates DNA synthesis. Preliminary results presented in this proposal suggest that CST functions in telomere replication and replication fork reinitiation. We propose that CST recruits DNA pol 1 to reinitiate DNA synthesis at sites of dificult-to-replicate DNA, which may stall DNA replication.
In aim 1, the role of CST in telomere replication will be investigated. Studies will then be extended to determine whether CST is required to maintain chromosomal fragile sites. Finally, the extent to which interactions between CST and the RecQ helicases, WRN and BLM, are required to maintain telomere and fragile site stability will be measured.
In aim 2, the mechanism by which CST promotes replication fork progression will be studied. Fork reinitiation after fork stalling and the levels of single-stranded DNA will be measured in CST-depleted cell lines. CST interactions with replication fork components and fork reinitiation factors will then be explored. Together, these studies will provide an understanding of how CST helps maintain genomic stability through difficult-to-replicate DNA.

Public Health Relevance

Each time a cell divides its DNA must be copied, or replicated, properly;if mistakes occur during replication, mutations can result which can lead to diseases, such as cancer and genetic disorders. Some cellular DNA is inherently difficult to replicate due to unusual DNA structure and may require the action of special proteins to promote proper replication. The proposed research will explore the role a newly discovered protein complex in replication of these difficult-to-replicate sites.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32GM097833-01
Application #
8123664
Study Section
Special Emphasis Panel (ZRG1-F08-E (20))
Program Officer
Carter, Anthony D
Project Start
2011-04-18
Project End
2013-04-17
Budget Start
2011-04-18
Budget End
2012-04-17
Support Year
1
Fiscal Year
2011
Total Cost
$51,326
Indirect Cost
Name
University of Cincinnati
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
041064767
City
Cincinnati
State
OH
Country
United States
Zip Code
45221
Wang, Feng; Stewart, Jason; Price, Carolyn M (2014) Human CST abundance determines recovery from diverse forms of DNA damage and replication stress. Cell Cycle 13:3488-98
Stewart, Jason A; Chaiken, Mary F; Wang, Feng et al. (2012) Maintaining the end: roles of telomere proteins in end-protection, telomere replication and length regulation. Mutat Res 730:12-9
Stewart, Jason A; Wang, Feng; Chaiken, Mary F et al. (2012) Human CST promotes telomere duplex replication and general replication restart after fork stalling. EMBO J 31:3537-49
Wang, Feng; Stewart, Jason A; Kasbek, Christopher et al. (2012) Human CST has independent functions during telomere duplex replication and C-strand fill-in. Cell Rep 2:1096-103