Mitochondrial base excision repair assays

TY - JOUR

T1 - Mitochondrial base excision repair assays

AU - Maynard, Scott

AU - de Souza-Pinto, Nadja C

AU - Scheibye-Knudsen, Morten

AU - Bohr, Vilhelm A

N1 - Published by Elsevier Inc.

PY - 2010/8/1

Y1 - 2010/8/1

N2 - 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 (POLγ 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.

AB - 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 (POLγ 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.

KW - Animals

KW - Cell Fractionation

KW - Cells, Cultured

KW - DNA Damage

KW - DNA Repair

KW - DNA Repair Enzymes

KW - DNA, Mitochondrial

KW - Deoxyguanosine

KW - Humans

KW - Mitochondria

KW - Polymerase Chain Reaction

U2 - 10.1016/j.ymeth.2010.02.020

DO - 10.1016/j.ymeth.2010.02.020

M3 - Review

C2 - 20188838

SN - 1046-2023

VL - 51

SP - 416

EP - 425

JO - Methods

JF - Methods

IS - 4

ER -