Free radical damage to DNA, which has been associated with carcinogenesis, the normal aging process, and neurodegeneration, produces a variety of lesions, including various oxidized abasic sites (OAS). These include 1'-oxidized, 4'-oxidized or fragmented deoxyribose residues, known products of agents such as H2O2, ionizing radiation, and bleomycin. Such damage have cytotoxic and mutagenic potential that might underlie the age and disease effects cited above, but OAS have been difficult to measure specifically, sensitively and quantitatively by existing methods. This project will capitalize on its initial success to continue development of and to apply new methods for such analysis, with an emphasis on the use of DNA repair enzymes to liberate specific damages for detection by advanced techniques of mass spectrometry. These methods will be used to explore the formation and repair of individual OAS in vivo. Abasic (AP) endonucleases are implicated in the repair of these damages, but this specifically has not been demonstrated in vivo. We will examine whether there are defects in repair of OAS in AP endonuclease-deficient bacteria, yeast and, if available, mammalian cells. The repairability in vitro and mutagenic potential in vivo of a specific OAS will be assessed using a synthetic, photosensitive 1'-t-butyl ketone derivative to target the damage in specific sequences. This work will be important for future work toward understanding the role of free-radical damage in aging, cancer, and neurodegeneration, and it will substantially improve the methodology for molecular analysis of this important class of oxidative DNA damage.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA071993-07
Application #
6513030
Study Section
Chemical Pathology Study Section (CPA)
Program Officer
Okano, Paul
Project Start
1996-07-25
Project End
2005-04-30
Budget Start
2002-05-01
Budget End
2003-04-30
Support Year
7
Fiscal Year
2002
Total Cost
$370,104
Indirect Cost
Name
Harvard University
Department
Genetics
Type
Schools of Public Health
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02115
Shao, Gang; Kautz, Roger; Peng, Shiqi et al. (2007) Calibration by NMR for quantitative analysis: p-toluenesulfonic acid as a reference substance. J Chromatogr A 1138:305-8
Sung, Jung-Suk; Demple, Bruce (2006) Analysis of base excision DNA repair of the oxidative lesion 2-deoxyribonolactone and the formation of DNA-protein cross-links. Methods Enzymol 408:48-64
Shimelis, Olga; Giese, Roger W (2006) Nuclease P1 digestion/high-performance liquid chromatography, a practical method for DNA quantitation. J Chromatogr A 1117:132-6
Chowdhury, Dipanjan; Beresford, Paul J; Zhu, Pengcheng et al. (2006) The exonuclease TREX1 is in the SET complex and acts in concert with NM23-H1 to degrade DNA during granzyme A-mediated cell death. Mol Cell 23:133-42
Sung, Jung-Suk; DeMott, Michael S; Demple, Bruce (2005) Long-patch base excision DNA repair of 2-deoxyribonolactone prevents the formation of DNA-protein cross-links with DNA polymerase beta. J Biol Chem 280:39095-103
Demple, Bruce; Sung, Jung-Suk (2005) Molecular and biological roles of Ape1 protein in mammalian base excision repair. DNA Repair (Amst) 4:1442-9
Fung, Hua; Demple, Bruce (2005) A vital role for Ape1/Ref1 protein in repairing spontaneous DNA damage in human cells. Mol Cell 17:463-70
Shao, Gang; Giese, Roger W (2004) Trace detection of glycolic acid by electrophore labeling gas chromatography-electron capture mass spectrometry. Anal Chem 76:3049-54
Shimelis, Olga; Zhou, Xiaojuan; Li, Guodong et al. (2004) Phenolic extraction of DNA from mammalian tissues and conversion to deoxyribonucleoside-5'-monophosphates devoid of ribonucleotides. J Chromatogr A 1053:143-9
Wong, Donny; Demple, Bruce (2004) Modulation of the 5'-deoxyribose-5-phosphate lyase and DNA synthesis activities of mammalian DNA polymerase beta by apurinic/apyrimidinic endonuclease 1. J Biol Chem 279:25268-75

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