title: An oxidative damage-specific endonuclease from rat liver mitochondria.
abstract: Reactive oxygen species have been shown to generate mutagenic lesions in DNA. One of the most abundant lesions in both nuclear and mitochondrial DNA is 7,8-dihydro-8-oxoguanine (8-oxoG). We report here the partial purification and characterization of a mitochondrial oxidative damage endonuclease (mtODE) from rat liver that recognizes and incises at 8-oxoG and abasic sites in duplex DNA. Rat liver mitochondria were purified by differential and Percoll gradient centrifugation, and mtODE was extracted from Triton X-100-solubilized mitochondria. Incision activity was measured using a radiolabeled double-stranded DNA oligonucleotide containing a unique 8-oxoG, and reaction products were separated by polyacrylamide gel electrophoresis. Gel filtration chromatography predicts mtODE's molecular mass to be between 25 and 30 kDa. mtODE has a monovalent cation optimum between 50 and 100 mM KCl and a pH optimum between 7.5 and 8. mtODE does not require any co-factors and is active in the presence of 5 mM EDTA. It is specific for 8-oxoG and preferentially incises at 8-oxoG:C base pairs. mtODE is a putative 8-oxoG glycosylase/lyase enzyme, because it can be covalently linked to the 8-oxoG oligonucleotide by sodium borohydride reduction. Comparison of mtODE's activity with other known 8-oxoG glycosylases/lyases and mitochondrial enzymes reveals that this may be a novel protein.
authors.affiliation: Laboratory of Molecular Genetics, NIA, National Institutes of Health, Baltimore, Maryland 21224, USA.
authors.forename: D L
authors.initials: DL
authors.lastname: Croteau
authors.forename: C M
authors.initials: CM
authors.lastname: ap Rhys
authors.forename: E K
authors.initials: EK
authors.lastname: Hudson
authors.forename: G L
authors.initials: GL
authors.lastname: Dianov
authors.forename: R G
authors.initials: RG
authors.lastname: Hansford
authors.forename: V A
authors.initials: VA
authors.lastname: Bohr
mesh_terms: D000818:Animals; D001483:Base Sequence; D002458:Cell Fractionation; D002850:Chromatography, Gel; D004260:DNA Repair; D004706:Endodeoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology* / Q000648:ultrastructure; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D018384:Oxidative Stress; D051381:Rats; D017208:Rats, Wistar; D013379:Substrate Specificity
chemical_list: D009838:Oligodeoxyribonucleotides; C024829:8-hydroxyguanine; D006147:Guanine; D004706:Endodeoxyribonucleases; C057799:3'-phosphoglycoaldehyde diesterase; C108945:mitochondrial oxidative damage endonuclease