Quantification of oxidative DNA modifications in mitochondria

Abstract

Specific repair endonucleases were used to quantify oxidative modifications in mitochondrial DNA (mtDNA) from rat liver and from porcine liver and kidney by means of a relaxation assay. In rat liver mitochondria the number of modifications sensitive to formamidopyrimidine-DNA glycosylase (FPG protein), which include 8-hydroxyguanine (8-oxo-7, 8-dihydro-guanine) residues, was only 0.8±0.2 per 10⁵ base pairs (bp). Even lower values were observed in porcine kidney (0.5±0.3 per 10⁵ bp) and liver (0.4±0.2 per 10⁵ bp). The numbers of sites of base loss (AP sites) sensitive to T4 endonuclease V and of 5,6-dihydropyrimidines sensitive to endonuclease III were less than 0.2 per 10⁵ bp in all cases. The data provide evidence that the steady-state levels of oxidative mtDNA modifications are low under physiological conditions, either because reactive oxygen species generated in the mitochondria are instantly inactivated or because of efficient DNA repair processes inside mitochondria.