An oxidative damage-specific endonuclease from rat liver mitochondria

J Biol Chem. 1997 Oct 24;272(43):27338-44. doi: 10.1074/jbc.272.43.27338.

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.

MeSH terms

  • Animals
  • Base Sequence
  • Cell Fractionation
  • Chromatography, Gel
  • DNA Repair*
  • Endodeoxyribonucleases / isolation & purification
  • Endodeoxyribonucleases / metabolism*
  • Guanine / analogs & derivatives
  • Male
  • Mitochondria, Liver / enzymology*
  • Mitochondria, Liver / ultrastructure
  • Molecular Weight
  • Oligodeoxyribonucleotides / chemistry
  • Oligodeoxyribonucleotides / metabolism
  • Oxidative Stress*
  • Rats
  • Rats, Wistar
  • Substrate Specificity

Substances

  • Oligodeoxyribonucleotides
  • 8-hydroxyguanine
  • Guanine
  • Endodeoxyribonucleases
  • 3'-phosphoglycoaldehyde diesterase
  • mitochondrial oxidative damage endonuclease