An oxidative damage-specific endonuclease from rat liver mitochondria

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 S-oxoG:C base pairs. mtODE is a putative S-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 glycosglases/lyases and mitochondrial enzymes reveals that this may be a novel protein.