Free radical-mediated DNA double-strand breaks (DSBs) induced by ionizing radiation, radiomimetic natural products, and oxidative stress have chemically modified termini such as 32-phosphoglycolates that must be removed before the break can be rejoined. In addition, accurate rejoining of these breaks requires replacement of fragmented nucleotides by gap filling on aligned DSB ends. Radiation-induced DSBs are also often accompanied by nearby oxidative base damage that can interfere with rejoining. DSBs induced by topoisomerase inhibitors have protein-linked termini that likewise must be removed for repair. The overall goal of the proposed project is to determine how these damaged DSB ends are resolved for repair by nonhomologous end joining, and how they affect the overall joining process. Candidate enzymes for processing damaged ends include tyrosyl-DNA phosphodiesterase (TDP1) and the DNA end-specific endonucleases Mre11, Artemis, Metnase and CtIP. Mouse and human cells with genetic defects in Artemis, Metnase, TDP1, Mre11 and combinations thereof will be augmented with siRNA knockdown to generate cells with various combinations of end-processing deficiencies. These cells will be subjected to cytotoxicity, cytogenetic and DSB repair assays, as well as a newly developed real-time PCR assay for measuring the persistence of blocked termini in cells. These studies will determine whether these repair factors provide alternative pathways for resolving blocked DNA termini, and the degree of overlap between them. Defined DSB substrates, bearing ends with 32-phosphoglycolates or oxidatively modified bases in various contexts, will be used to determine the specificities of Artemis and Metnase in trimming damaged ends. The efficiency with which the resulting trimmed ends progress to the gap filling and ligation steps in cell extracts will be determined. Tolerance for damaged bases in gap-filling on aligned DSB ends by DNA polymerase;will be determined, as well as the possible cooperation and competition between polymerase;and Artemis in resolving different types of damaged DNA termini.

Public Health Relevance

Because radiotherapy and some types of chemotherapy kill tumor cells by inducing DNA double- strand breaks, enzymes that process damage at the ends of such breaks represent potential therapeutic targets that could be exploited to improve efficacy and therapeutic index. Moreover, some repair enzymes delete segments of normal DNA in the process of removing damage from the ends, and therefore the accuracy of repair can depend on the specific repair enzymes used, which is the focus of the proposed research. Because inaccurate double-strand break repair can lead to genomic alterations that promote malignancy, it is essential in terms of cancer prevention to understand how the choice of repair enzymes and repair systems is made by the cell for specific types of double-strand breaks.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA040615-27
Application #
8469394
Study Section
Cancer Etiology Study Section (CE)
Program Officer
Pelroy, Richard
Project Start
1985-08-01
Project End
2015-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
27
Fiscal Year
2013
Total Cost
$232,974
Indirect Cost
$77,138
Name
Virginia Commonwealth University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
105300446
City
Richmond
State
VA
Country
United States
Zip Code
23298
Menon, Vijay; Povirk, Lawrence (2014) Involvement of p53 in the repair of DNA double strand breaks: multifaceted Roles of p53 in homologous recombination repair (HRR) and non-homologous end joining (NHEJ). Subcell Biochem 85:321-36
Akopiants, Konstantin; Mohapatra, Susovan; Menon, Vijay et al. (2014) Tracking the processing of damaged DNA double-strand break ends by ligation-mediated PCR: increased persistence of 3'-phosphoglycolate termini in SCAN1 cells. Nucleic Acids Res 42:3125-37
Mohapatra, Susovan; Yannone, Steven M; Lee, Suk-Hee et al. (2013) Trimming of damaged 3' overhangs of DNA double-strand breaks by the Metnase and Artemis endonucleases. DNA Repair (Amst) 12:422-32
Menon, Vijay R; Peterson, Erica J; Valerie, Kristoffer et al. (2013) Ligand modulation of a dinuclear platinum compound leads to mechanistic differences in cell cycle progression and arrest. Biochem Pharmacol 86:1708-20
Dever, Seth M; Golding, Sarah E; Rosenberg, Elizabeth et al. (2011) Mutations in the BRCT binding site of BRCA1 result in hyper-recombination. Aging (Albany NY) 3:515-32
Mohapatra, Susovan; Kawahara, Misako; Khan, Imran S et al. (2011) Restoration of G1 chemo/radioresistance and double-strand-break repair proficiency by wild-type but not endonuclease-deficient Artemis. Nucleic Acids Res 39:6500-10
Khalil, Ashraf; Morgan, Rhiannon N; Adams, Bret R et al. (2011) ATM-dependent ERK signaling via AKT in response to DNA double-strand breaks. Cell Cycle 10:481-91
Zhou, Rui-Zhe; Akopiants, Konstantin; Povirk, Lawrence F (2010) Patching and single-strand ligation in nonhomologous DNA end joining despite persistence of a closely opposed 3'-phosphoglycolate-terminated strand break. Radiat Res 174:274-9
Taylor, Elaine M; Cecillon, Sophie M; Bonis, Antonio et al. (2010) The Mre11/Rad50/Nbs1 complex functions in resection-based DNA end joining in Xenopus laevis. Nucleic Acids Res 38:441-54
Bebenek, Katarzyna; Garcia-Diaz, Miguel; Zhou, Rui-Zhe et al. (2010) Loop 1 modulates the fidelity of DNA polymerase lambda. Nucleic Acids Res 38:5419-31

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