Abstract

We have developed assays to detect oxidative lesions in specific genes and to quantitate their formation and repair. Oxidative DNA damage is generated by several different approaches including hydrogen peroxide, acridine orange, X-irradiation, irradiation with methylene blue. The main lesion which we examine is 8-OH guanosine which can be detected by use of the Fpg glycosylase. This enzyme creates strand breaks in DNA at sites of the lesions, and the single stranded DNA can then be resolved on alkaline gels. We find that 8-OH guanosine is rapidly repaired in active genes and in mitochondrial DNA. We have developed an in vitro assay for DNA nicking activity in mitochondrial extracts from rats. We have identified a protein from mitochondria which is able to incise an oligo containing a site specific 8-oxoguanine adduct. Unlike the bacterial enzyme which cleaves both 3' and 5' to the lesion, the mitochondrial enzyme incises exclusively on the 3' side of the lesion. We have finalized our chromatography steps and have begun the physical characterization of the enzyme.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Intramural Research (Z01)
Project #
1Z01AG000727-04
Application #
2565751
Study Section
Special Emphasis Panel (LMG)
Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
1996
Total Cost
Indirect Cost

  Institution

Name
National Institute on Aging
Department
Type
DUNS #
City
State
Country
United States
Zip Code

  Publications

Baptiste, Beverly A; Katchur, Steven R; Fivenson, Elayne M et al. (2018) Enhanced mitochondrial DNA repair of the common disease-associated variant, Ser326Cys, of hOGG1 through small molecule intervention. Free Radic Biol Med 124:149-162
Gredilla, Ricardo; Bohr, Vilhelm A; Stevnsner, Tinna (2010) Mitochondrial DNA repair and association with aging--an update. Exp Gerontol 45:478-88
Kirkali, Güldal; de Souza-Pinto, Nadja C; Jaruga, Pawel et al. (2009) Accumulation of (5'S)-8,5'-cyclo-2'-deoxyadenosine in organs of Cockayne syndrome complementation group B gene knockout mice. DNA Repair (Amst) 8:274-8
de Souza-Pinto, Nadja C; Maynard, Scott; Hashiguchi, Kazunari et al. (2009) The recombination protein RAD52 cooperates with the excision repair protein OGG1 for the repair of oxidative lesions in mammalian cells. Mol Cell Biol 29:4441-54
Maynard, Scott; Schurman, Shepherd H; Harboe, Charlotte et al. (2009) Base excision repair of oxidative DNA damage and association with cancer and aging. Carcinogenesis 30:2-10
Schurman, Shepherd H; Hedayati, Mohammad; Wang, ZhengMing et al. (2009) Direct and indirect roles of RECQL4 in modulating base excision repair capacity. Hum Mol Genet 18:3470-83
Stevnsner, Tinna; Muftuoglu, Meltem; Aamann, Maria Diget et al. (2008) The role of Cockayne Syndrome group B (CSB) protein in base excision repair and aging. Mech Ageing Dev 129:441-8
Muftuoglu, Meltem; Kusumoto, Rika; Speina, Elzbieta et al. (2008) Acetylation regulates WRN catalytic activities and affects base excision DNA repair. PLoS ONE 3:e1918
Aune, Gregory J; Takagi, Kazutaka; Sordet, Olivier et al. (2008) Von Hippel-Lindau-coupled and transcription-coupled nucleotide excision repair-dependent degradation of RNA polymerase II in response to trabectedin. Clin Cancer Res 14:6449-55
Harrigan, Jeanine A; Fan, Jinshui; Momand, Jamil et al. (2007) WRN exonuclease activity is blocked by DNA termini harboring 3'obstructive groups. Mech Ageing Dev 128:259-66

Showing the most recent 10 out of 46 publications