The goals of this project are to define the mechanisms and extent of nucleotide expression repair (NER) inhibition at telomeres, and the impact of unrepaired DNA lesions on telomere structure and function. Telomeres at chromosome ends are essential for genome stability and sustained cell proliferation. Telomeric DNA is highly susceptible to photoproduct formation caused by ultraviolet (UV) light, which are removed by NER in the bulk genome. This proposal will test the hypothesis that telomere binding proteins prevent DNA lesion removal at telomeres by inhibiting the enzymatic activities of NER enzymes. Our preliminary studies show that photoproducts induce telomere loss and aberrations by interfering with telomere replication, which is consistent with a deficiency in lesion removal at telomeres. We further show that a telomeric protein inhibits the catalytic activity of a nuclease required for NER in vitro.
Aim 1 will compare endpoints of telomeric damage and dysfunction in UVC irradiated NER proficient- and deficient- cells to establish how photoproducts impact individual telomeres. We will measure photoproduct repair rates in telomeres, compared to the bulk genome, using an innovative assay that quantifies photoproducts in telomeres isolated from UVC exposed human cells.
Aim 2 will test for recruitment of key NER proteins to damaged telomeric regions, compared with non-telomeric regions, in cell nuclei using fluorescent protein tags and live cell imaging. Laser micro-irradiatin will be used to generate photoproducts and bulky adducts at define regions in the cell nucleus.
Aim 3 will examine how telomeric proteins modulate various enzymatic steps in the NER process. NER will be examined in vitro using cell extracts on defined telomeric and non-telomeric substrates in the presence of individual telomeric proteins or the complete telomeric protein complex. NER is required for removing a wide variety of DNA lesions generated by environmental genotoxicants and anti-cancer drugs. This project will fill a significant void in our understanding of how telomeres evade NER, and how unrepaired DNA lesions alter telomere structure and function. This knowledge will be highly valuable for developing new strategies that 1) preserve telomeres to mitigate the effects of environmental genotoxicant exposures or conversely, that 2) inhibit global genome NER to sensitize malignant cells for killing by anti-cancer genotoxic drugs.

Public Health Relevance

Telomeres at chromosome ends prevent genomic instability which contributes to carcinogenesis. Nucleotide excision repair removes bulky DNA lesions caused by environmental and anti-cancer DNA damaging agents. Understanding how nucleotide excision repair is suppressed at telomeres will aid the development of new therapeutic strategies that preserve telomeres in healthy cells after genotoxic exposures, and that conversely, extend NER suppression to the bulk genome in cancer cells to enhance killing by anti-cancer drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES022944-03
Application #
8854084
Study Section
Cancer Genetics Study Section (CG)
Program Officer
Heacock, Michelle
Project Start
2013-09-01
Project End
2016-05-31
Budget Start
2015-06-01
Budget End
2016-05-31
Support Year
3
Fiscal Year
2015
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Public Health & Prev Medicine
Type
Graduate Schools
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Barnes, Ryan P; Fouquerel, Elise; Opresko, Patricia L (2018) The impact of oxidative DNA damage and stress on telomere homeostasis. Mech Ageing Dev :
Fouquerel, Elise; Opresko, Patricia L (2017) Convergence of The Nobel Fields of Telomere Biology and DNA Repair. Photochem Photobiol 93:229-237
Orenstein, Alexander; Berlyoung, April S; Rastede, Elizabeth E et al. (2017) ?PNA FRET Pair Miniprobes for Quantitative Fluorescent In Situ Hybridization to Telomeric DNA in Cells and Tissue. Molecules 22:
Zhou, Jia; Chan, Jany; Lambelé, Marie et al. (2017) NEIL3 Repairs Telomere Damage during S Phase to Secure Chromosome Segregation at Mitosis. Cell Rep 20:2044-2056
Fouquerel, Elise; Parikh, Dhvani; Opresko, Patricia (2016) DNA damage processing at telomeres: The ends justify the means. DNA Repair (Amst) 44:159-168
Kaur, Parminder; Wu, Dong; Lin, Jiangguo et al. (2016) Enhanced electrostatic force microscopy reveals higher-order DNA looping mediated by the telomeric protein TRF2. Sci Rep 6:20513
Garcia-Exposito, Laura; Bournique, Elodie; Bergoglio, Valérie et al. (2016) Proteomic Profiling Reveals a Specific Role for Translesion DNA Polymerase ? in the Alternative Lengthening of Telomeres. Cell Rep 17:1858-1871
Lin, Jiangguo; Countryman, Preston; Chen, Haijiang et al. (2016) Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing. Nucleic Acids Res 44:6363-76
Fouquerel, Elise; Lormand, Justin; Bose, Arindam et al. (2016) Oxidative guanine base damage regulates human telomerase activity. Nat Struct Mol Biol 23:1092-1100
Parikh, Dhvani; Fouquerel, Elise; Murphy, Connor T et al. (2015) Telomeres are partly shielded from ultraviolet-induced damage and proficient for nucleotide excision repair of photoproducts. Nat Commun 6:8214

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