Free radical damage to DNA, which has been associated with carcinogenesis, the normal aging process, and neurodegeneration, produces a variety of lesions. Many of these damages are various abasic lesions (sites of base loss). These include 1 -oxidized, 4 -oxidized or fragmented deoxyribose residues, known products of agents such as H2O2, ionizing radiation and bleomycin. Such damages have cytotoxic and mutagenic potential that might underlie the age and disease effects cited above, but these oxidized deoxyribose lesions have been difficult to measure specifically, sensitively and quantitatively by existing methods. This project proposes to develop new methods for such analysis, with an emphasis on the use of DNA repair enzymes to liberate specific damages for analysis by advanced techniques of mass spectrometry and capillary electrophoresis. These methods will be used to explore the formation and repair of individual oxidized abasic sites in vivo. Abasic (AP) endonucleases have been implicated in the repair of these damages, but this specificity has not been demonstrated in vivo. Key proposed studies include analyzing chromosomal DNA to determine the specific role of E. coli exonuclease III in repairing H2O2-induced DNA damage, and of E. coli endonuclease IV and yeast Apnl in the repair of oxidized abasic sites. The ability of human Ape endonuclease to correct specific repair defects in mutant strains of E. coli and yeast will be assessed by the novel methods, as will mutated forms of endonuclease IV and Apnl. Not only will this work be important for future work toward understanding the role of free-radical damage in aging, cancer, and neurodegeneration, but it will also provide quantum improvements in the methodology for molecular analysis of an important class of oxidative DNA damage.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA071993-04
Application #
2895685
Study Section
Chemical Pathology Study Section (CPA)
Program Officer
Okano, Paul
Project Start
1996-07-25
Project End
2000-04-30
Budget Start
1999-05-01
Budget End
2000-04-30
Support Year
4
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Harvard University
Department
Other Basic Sciences
Type
Schools of Public Health
DUNS #
082359691
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
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
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

Showing the most recent 10 out of 28 publications