Quantification of oxidative DNA modifications in mitochondria

Specific repair endonucleases were used to quantify oxidativemodifications in mitochondrial DNA (mtDNA) from rat liver andfrom porcine liver and kidney by means of a relaxation assay.In rat liver mitochondria the number of modifications sensitiveto formamidopyrimidine-DNA glycosylase (FPG protein), whichinclude 8-hydroxyguanine (8-oxo-7, 8-dihydro-guanine) residues,was only 0.8±0.2 per 10⁵ base pairs (bp). Even lowervalues were observed in porcine kidney (0.5±0.3 per 10⁵bp) and liver (0.4±0.2 per 10⁵ bp). The numbers of sitesof base loss (AP sites) sensitive to T4 endonuclease V and of5,6-dihydropyrimidines sensitive to endonuclease III were lessthan 0.2 per 10⁵ bp in all cases. The data provide evidencethat the steady-state levels of oxidative mtDNA modificationsare low under physiological conditions, either because reactiveoxygen species generated in the mitochondria are instantly inactivatedor because of efficient DNA repair processes inside mitochondria.     

Singlet oxygen as an ultimately reactive species in Salmonella typhimurium DNA damage induced by methylene blue/visible light

The specific recognition of various DNA modifications by repairenzymes is exploited for the analysis of DNA damage inducedby visible light in the presence of methylene blue in Salmonellatyphimurium. The relative frequencies of various endonudease-sensitivesites and strand breaks are determined in the plasmid pAQl ofthe treated bacteria and are compared with those observed afterexposure of isolated DNA to various conditions. This comparisonof damage profiles indicates that the cellular DNA damage byillumination in the presence of methylene blue is caused predominantlyby the direct action of singlet oxygen. Indirect mechanisms,e.g. involving a generation of superoxide and hydroxyl radicalsor the activation of cellular nucleases, do not contribute verymuch. The damage is dominated by base modifications. These aresubject to an efficient repair that is not mediated by uvrABCproteins and therefore most probably involves recognition byspecific glycosylases. Revertant frequencies observed underthese conditions in the strains TA1535, TA100, TA2638 and TA104indicate a pronounced mutagenicity of the lesions induced. Onthe other hand, the DNA damage does not contribute significantlyto the cytotoxicity caused by the treatment as an excision repairdeficiency (uvrB) has no influence on cell killing.     

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