Nitric oxide-induced damage to mtDNA and its subsequent repair

Mutations in mitochondrial DNA (mtDNA) have recently been associated with a variety of human diseases. One potential DNA-damaging agent to which cells are continually exposed that could be responsible for some of these mutati ons is nitric oxide (NO). To date, little information has been forthcoming concerning the damage caused by this gas to mtDNA. Therefore, this study wa s designed to investigate damage to mtDNA induced by NO and to evaluate its subsequent repair. Normal human fibroblasts were exposed to NO produced by the rapid decomposition of 1-propanamine,3-(2-hydroxy-2-nitroso-1-propylhy drazinc) (PAPA NONOate) and the resultant damage to mtDNA was determined by quantitative Southern blot analysis. This gas was found to cause damage to mtDNA that was alkali-sensitive. Treatment of the DNA with uracil-DNA glyc osylase or 3-methyladenine DNA glycosylase failed to reveal additional dama ge, indicating that most of the lesions produced were caused by the deamina tion of guanine to xanthine. Studies using ligation-mediated PCR supported this finding. When a 200 bp sequence of mtDNA from cells exposed to NO was analyzed, guanine was found to be the predominantly damaged base. However, there also was damage to specific adenines. No lesions were observed at pyr imidine sites. The nucleotide pattern of damage induced by NO was different from that produced by either a reactive oxygen species generator or the me thylating chemical, methylnitrosourea. Most of the lesions produced by NO w ere repaired rapidly. However, there appeared to be a subset of lesions whi ch were repaired either slowly or not at all by the mitochondria.