Enhanced mitochondrial DNA repair and cellular survival after oxidative stress by targeting the human 8-oxoguanine glycosylase repair enzyme to mitochondria

Oxidative damage to mitochondrial DNA (mtDNA) has been implicated as a caus ative factor in many disease processes and in aging. We have recently disco vered that different cell types vary in their capacity to repair this damag e, and this variability correlates with their ability to withstand oxidativ e stress. To explore strategies to enhance repair of oxidative lesions in m tDNA, we have constructed a vector containing a mitochondrial transport seq uence upstream of the sequence for human 8-oxoguanine DNA glycosylase. This enzyme is the glycosylase/AP lyase that participates in repair of purine l esions, such as 8-oxoguanine. Western blot analysis confirmed that this rec ombinant protein was targeted to mitochondria, Enzyme activity assays showe d that mitochondrial extracts from cells transfected with the construct had increased enzyme activity compared with cells transfected with vector only , whereas nuclear enzyme activity was not changed. Repair assays showed tha t there was enhanced repair of oxidative lesions in mtDNA, Additional studi es revealed that this augmented repair led to enhanced cellular viability a s determined by reduction of the tetrazolium compound to formazan, trypan b lue dye exclusion, and clonogenic assays. Therefore, targeting of DNA repai r enzymes to mitochondria may be a viable approach for the protection of ce lls against some of the deleterious effects of oxidative stress.