Mitochondrial base excision repair assays.
The main source of mitochondrial DNA (mtDNA) damage is reactive oxygen species (ROS) generated during normal cellular metabolism. The main mtDNA lesions generated by ROS are base modifications, such as the ubiquitous 8-oxoguanine (8-oxoG) lesion; however, base loss and strand breaks may also occur. Many human diseases are associated with mtDNA mutations and thus maintaining mtDNA integrity is critical. All of these lesions are repaired primarily by the base excision repair (BER) pathway. It is now known that mammalian mitochondria have BER, which, similarly to nuclear BER, is catalyzed by DNA glycosylases, AP endonuclease, DNA polymerase (POLgamma in mitochondria) and DNA ligase. This article outlines procedures for measuring oxidative damage formation and BER in mitochondria, including isolation of mitochondria from tissues and cells, protocols for measuring BER enzyme activities, gene-specific repair assays, chromatographic techniques as well as current optimizations for detecting 8-oxoG lesions in cells by immunofluorescence. Throughout the assay descriptions we will include methodological considerations that may help optimize the assays in terms of resolution and repeatability.
Maynard
Scott
S
Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, MD 21236, USA.
de Souza-Pinto
Nadja C
NC
Scheibye-Knudsen
Morten
M
Bohr
Vilhelm A
VA
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D002458:Cell Fractionation; D002478:Cells, Cultured; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics / Q000502:physiology; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000235:genetics / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000032:analysis; D006801:Humans; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction
D004272:DNA, Mitochondrial; D000080242:8-Hydroxy-2'-Deoxyguanosine; D045643:DNA Repair Enzymes; D003849:Deoxyguanosine