Mitochondrial respiration provides a major source of energy for eukaryotic cells. However, the energy-producing processes also generate reactive oxygen species, which in turn damage mitochondrial DNA found in the mitochondrial matrix. Due to its locale, mitochondrial DNA is more susceptible to oxidative damage than nuclear DNA. While mitochondria do have some DNA repair capabilities, particularly base excision repair, oxidative damage persists in mitochondrial DNA. Correlations have been demonstrated between increasing age and increased levels of oxidative damage and mitochondrial DNA mutations. The current experiments were designed to begin to more directly delineate the role oxidative damage in mitochondrial DNA plays in aging. The mouse myoblast cell line, C2C12, was transfected with vectors, which express formamidopyrimidine—DNA glycosylase-mycfusion protein (Fpg-myc) and which contain either a mitochondrial or nuclear localization signal. Positive transfectants display expression offpgat the mRNA level and exhibit an increase in Fpg activity in a whole-cell protein extract using a Fpg activity assay. Immunofluorescence analyses confirm that the transfected vectors have Fpg-mycappropriately targeted to mitochondria or nuclei. These cell lines with specifically targeted Fpg-mycexpression provide the tools to test the effects of increasing the levels of a DNA glycosylase in mitochondria and nuclei on oxidative damage in DN
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