Repair of oxidative damage within the mitochondrial DNA of RINr 38 cells

A growing body of evidence suggests that a variety of chronic diseases, including cancer and diabetes, are associated with damage to mitochondrial DNA. Since mitochondria are constantly exposed to high levels of reactive oxygen species, it is likely that oxidative damage to mitochondrial DNA may be responsible for some of these maladies. To determine whether mitochondria can repair this damage, a quantitative Southern blot technique was utilized to identify repair in specific DNA fragments. A 10.8-kilobase mitochondrial restriction fragment was studied employing a probe containing the entire mouse mitochondrial genome. Alloxan was employed to generate oxygen radicals. Insulinoma cells were exposed to alloxan for 1 h, and total cellular DNA was isolated immediately or after intervals of up to 8 h. Alkali treatment was used to identify abasic sites and sugar lesions, endonuclease III was used to identify lesions associated with thymine and cytosine damage, and formamidopyrimidine-DNA glycosylase was employed to recognize formamidopyrimidines and 8-oxoguanines in DNA. The results showed that all forms of damage studied were repaired by 4 h, indicating that mitochondria are able to efficiently repair damage to their DNA caused by reactive oxygen species.