Mitochondrial base excision repair assays

Abstract

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.

Fig. 1

Summary of DNA repair pathways characterized in the nucleus and mitochondria. All pathways are well characterized in the nucleus. There is no evidence for nucleotide excision repair or non homologous end joining (NHEJ) in the mitochondrial. There is has been homologous replication (HR) repair activity detected in the mitochondria of the cells of some animal species under some conditions, but it is not determined as to how commonly this pathway indeed occurs in mitochondria.

Fig. 2

Endogenous oxidative damage is overestimated in mtDNA due to oxidative lesions generated from the mtDNA purification process. Fpg/Southern blot analysis was used to measure 8-oxoG levels in nDNA as well as in mtDNA with and without mitochondrial isolation; the crude homogenate and purified mtDNA was obtained from rat liver. Levels of DNA damage detected from nuclear and mitochondrial sequences in DNA isolated from crude homogenates were not significantly different. The endogenous levels based on DNA from isolated mitochondria were approximately three fold higher. Adpated from Anson et al. [43]

Fig. 3

The principle behind the Southern blot gene-specific damage and repair assay. A DNA repair enzyme is used as a tool to nick a restricted DNA fragment at the site of damage. In a denaturing agarose gel, the cleaved strand migrates further than the full length restriction fragment. The band is detected by Southern blot. Obtained from Anson et al. [63].

Fig 4

Use of gene-specific probes and Southern blotting to demonstrate efficient repair of 8-oxoG in mtDNA. (A) Regions of the Chinese hamster ovary (CHO) genome analyzed. K, KpnI. Horizontal bars below the gene-maps indicate the probes used. The DHFR gene was probed for by pMB5; the non-transcribed region was probed for by CS14; the rDNA was probed for by pA_BB which recognizes a 9 kb fragment containing the 5.8S and 28S region; the probing of the mtDNA by pCRII visualizes a 16 kb fragment. The map is based on the sequence known for guinea pig mitochondrial genome. (B) Repair of 8-oxoG in the genes probes for in part (A) was measured. KpnI digested genomic DNA (1 μg) from RO treated cells, and mock treated cells (control), was isolated at various time points after damage induction. Before Southern blot analysis DNA was either mock treated or treated with FPG (−/+). Representative autoradiograms of Southern blots are shown in the top panel, for the above probed genes. In the bottom panel quantification of the bands in the Southern blot is presented as percent repair of photoactivated RO induced 8-oxoG in the four different regions of the genome investigated. The data presented are the average +/− standard deviation from five biological experiments with several gels from each experiment. Adapted from Thorslund et al. [64].

Fig 4

Use of gene-specific probes and Southern blotting to demonstrate efficient repair of 8-oxoG in mtDNA. (A) Regions of the Chinese hamster ovary (CHO) genome analyzed. K, KpnI. Horizontal bars below the gene-maps indicate the probes used. The DHFR gene was probed for by pMB5; the non-transcribed region was probed for by CS14; the rDNA was probed for by pA_BB which recognizes a 9 kb fragment containing the 5.8S and 28S region; the probing of the mtDNA by pCRII visualizes a 16 kb fragment. The map is based on the sequence known for guinea pig mitochondrial genome. (B) Repair of 8-oxoG in the genes probes for in part (A) was measured. KpnI digested genomic DNA (1 μg) from RO treated cells, and mock treated cells (control), was isolated at various time points after damage induction. Before Southern blot analysis DNA was either mock treated or treated with FPG (−/+). Representative autoradiograms of Southern blots are shown in the top panel, for the above probed genes. In the bottom panel quantification of the bands in the Southern blot is presented as percent repair of photoactivated RO induced 8-oxoG in the four different regions of the genome investigated. The data presented are the average +/− standard deviation from five biological experiments with several gels from each experiment. Adapted from Thorslund et al. [64].