HOMOGENOUS REPAIR OF SINGLET OXYGEN-INDUCED DNA-DAMAGE IN DIFFERENTIALLY TRANSCRIBED REGIONS AND STRANDS OF HUMAN MITOCHONDRIAL-DNA

Photoactivated methylene blue was used to damage purified DNA and the mitochondrial DNA (mtDNA) of human fibroblasts in culture. The primary product of this reaction is the DNA lesion 7-hydro-8-oxo-deoxyguanosi ne (8-oxo-dG). The DNA damage was quantitated using Escherichia coli f ormamido-pyrimidine DNA glycosylase (Fpg) in a gene-specific damage an d repair assay, Assay conditions were refined to give incision at ail enzyme-sensitive sites with minimal non-specific cutting, Cultured fib roblasts were exposed to photoactivated methylene blue under condition s that would produce an average of three oxidative lesions per double- stranded mitochondrial genome. Within 9 h, 47% of this damage had been removed by the cells, This removal was due to repair rather than to r eplication, cell loss or degradation sf damaged genomes, The rate of r epair was measured in both DNA strands of the frequently transcribed r ibosomal region of the mitochondrial genome and in both strands of the non-ribosomal region, Fpg-sensitive alkali-resistant oxidative base d amage was efficiently removed from human mtDNA with no differences in the rate of repair between strands or between two different regions of the genome that differ substantially with regard to transcriptional a ctivity.