Defective repair of oxidative damage in the mitochondrial DNA of a xeroderma pigmentosum group A cell line.

Recent evidence has linked mitochondrial DNA (mtDNA) damage to several disease processes,including cancer and aging. An important source of such damage is reactive oxygen species. These molecules can be generated endogenously via the electron transport system or may arise from a host of exogenous sources. It has been reported that extracts from cells of individuals with xeroderma pigmentosum group A (XP-A) do not repair some types of oxidative DNA damage. The current experiments were designed to determine whether there is a correlation between the inadequate repair of oxidatively damaged nuclear DNA in XP-A cells and the capacity of such cells to repair similar damage to their mtDNA. The ability of karyotypically normal human fibroblasts (WI-38) and XP-A fibroblasts to repair alloxan-generated oxidative damage to nuclear and mtDNA was assessed using a quantitative Southern blot method in conjunction with the repair enzymes endonuclease III and formamidopyrimidine DNA glycosylase. The data indicate that both nuclear and mtDNA repair of each damage type investigated is more efficient in the WI-38 cells. These findings suggest a similarity between the process(es) used to repair oxidative damage to nuclear and mtDNA in that both are inhibited by the defect in XP-A.

Repair of oxidative damage in nuclear DNA sequences with different transcriptional activities.

This study was designed to investigate the repair of oxidative damage in nuclear DNA sequences with different transcriptional activities. Chinese hamster ovary (CHO) cells were treated with the oxygen radical generator hypoxanthine/xanthine oxidase (Hyp/XO). Damage and repair were evaluated in 14-kb restriction fragments containing either the DHFR gene, a 3'-non-transcribed flanking region, or the c-fos gene using a quantitative Southern blot technique. Damage to the sugar-phosphate backbone and abasic sites were detected by measuring their lability in alkali conditions. Lesions in DNA bases were identified using the bacterial repair enzyme endonuclease III, which predominantly recognizes damage to thymines and cytosines, and formamidopyrimidine-DNA glycosylase, which recognizes 8-oxoguanine and purines with fractured imidazole rings. The results showed that similar amounts of all types of oxidative damage were produced in both the transcribed and non-transcribed sequences following a 1-h exposure to the radical generator. Repair in all sequences was rapid, with approximately 60% removal of lesions observed by 1 h. Therefore, within these sequences, the repair of oxidative lesions is much faster than that of other types of damage, such as those induced by alkylating toxins and UV irradiation, and the repair is not affected appreciably by transcriptional status.

Repair of mitochondrial DNA damage induced by bleomycin in human cells.

Damage to mitochondrial DNA (mtDNA) has recently been associated with a variety of human diseases including cancer, diabetes mellitus, and aging. The mechanisms by which the mitochondria respond to DNA damage are of prime importance in understanding how damage can persist and cause disease. Here we demonstrate the repair of mitochondrial DNA damage induced by the naturally occurring, radiomimetic drug bleomycin. WI-38 cells were first permeabilized using 20 micrograms/ml lysophosphatidylcholine in order to increase the intracellular concentration of bleomycin. Dose response studies with the permeabilized cells showed that a concentration of 5 micrograms/ml bleomycin given for 30 min caused sufficient DNA damage for repair studies. Following treatment with this concentration of bleomycin, repair of mtDNA damage was found to be about 80% by 2 h. However, after 4 h no additional repair was observed. The results indicate that there is an efficient DNA repair system in human mitochondria for some types of damage caused by bleomycin. However, there is a component of damage caused by this agent that either is not repaired or is removed at a much slower rate.

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.

Mapping frequencies of endogenous oxidative damage and the kinetic response to oxidative stress in a region of rat mtDNA.

Genomic DNA is constantly being damaged and repaired and our genomes exist at lesion equilibrium for damage created by endogenous mutagens. Mitochondrial DNA (mtDNA) has the highest lesion equilibrium frequency recorded; presumably due to damage by H2O2 and free radicals generated during oxidative phosphorylation processes. We measured the frequencies of single strand breaks and oxidative base damage in mtDNA by ligation-mediated PCR and a quantitative Southern blot technique coupled with digestion by the enzymes endonuclease III and formamidopyrimidine DNA glycosylase. Addition of 5 mM alloxan to cultured rat cells increased the rate of oxidative base damage and, by several fold, the lesion frequency in mtDNA. After removal of this DNA damaging agent from culture, the single strand breaks and oxidative base damage frequency decreased to levels slightly below normal at 4 h and returned to normal levels at 8 h, the overshoot at 4 h being attributed to an adaptive up-regulation of mitochondrial excision repair activity. Guanine positions showed the highest endogenous lesion frequencies and were the most responsive positions to alloxan-induced oxidative stress. Although specific bases were consistently hot spots for damage, there was no evidence that removal of these lesions occurred in a strand-specific manner. The data reveal non-random oxidative damage to several nucleotides in mtDNA and an apparent adaptive, non-strand selective response for removal of such damage. These are the first studies to characterize oxidative damage and its subsequent removal at the nucleotide level in mtDNA.

Mapping oxidative DNA damage using ligation-mediated polymerase chain reaction technology.

Reactive oxygen species induce a pharmacopoeia of oxidized bases in DNA. DNA can be cleaved at most of the sites of these modified bases by digestion with a combination of two base excision repair glycosylases from Escherichia coli, Fpg glycosylase, and endonuclease III. The frequency of the resulting glycosylase-dependent 5'-phosphoryl ends can be mapped at nucleotide resolution along a sequencing gel autoradiogram by a genomic sequencing technique, ligation-mediated polymerase chain reaction (LMPCR). In cultured rat cells, the frequency of endogenous oxidized bases in mitochondrial DNA is sufficiently high, about one oxidized base per 100 kb, to be directly mapped from 0.1 microg of total cellular DNA preparations by LMPCR. Nuclear DNA has a lower frequency of endogenous oxidative base damage which cannot be mapped from 1-microg preparations of total cellular DNA. Preparative gel electrophoresis of the PGK1 and p53 genes from 300 microg of restriction endonuclease-digested genomic DNA showed a 25-fold enrichment for the genes and, after endonuclease digestion followed by LMPCR, gave sufficient signal to map the frequency of oxidized bases from human cells treated with 50 microM H2O2.