Quantification of oxidative DNA modifications in mitochondria.
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-dihydroguanine) residues, was only 0.8 +/- 0.2 per 10(5) base pairs (bp). Even lower values were observed in porcine kidney (0.5 +/- 0.3 per 10(5) bp) and liver (0.4 +/- 0.2 per 10(5) 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 10(5) 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.
Hegler
J
J
Institute of Pharmacology and Toxicology, University of Würzburg, Germany.
Bittner
D
D
Boiteux
S
S
Epe
B
B
D000818:Animals; D004249:DNA Damage; D004260:DNA Repair; D004272:DNA, Mitochondrial / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D043382:DNA-Formamidopyrimidine Glycosylase; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004587:Electrophoresis, Agar Gel; D004706:Endodeoxyribonucleases / Q000378:metabolism; D005260:Female; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D008297:Male; D008928:Mitochondria / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000378:metabolism*; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D016877:Oxidants / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013552:Swine; D014764:Viral Proteins
D004272:DNA, Mitochondrial; D016877:Oxidants; D014764:Viral Proteins; C024829:8-hydroxyguanine; D006147:Guanine; D004706:Endodeoxyribonucleases; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer); D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase