Use of a molecular beacon to track the activity of base excision repair protein OGG1 in live cells.
An abundant form of DNA damage caused by reactive oxygen species is 8-oxo-7,8-dihydroguanine for which the base excision repair protein 8-oxoguanine-DNA glycosylase 1 (OGG1) is a major repair enzyme. To assess the location and intracellular activity of the OGG1 protein in response to oxidative stress, we have utilised a fluorescence-quench molecular beacon switch containing a 8-oxo-dG:C base pair and a fluorescent and quencher molecule at opposite ends of a hairpin oligonucleotide. Oxidative stress was induced by treatment with potassium bromate. Flow cytometry demonstrated a concentration-dependent increase in the activity of OGG1 that was detected by the fluorescence produced when the oligonucleotide was cleaved in the cells treated with potassium bromate. This signal is highly specific and not detectable in OGG1 knock out cells. Induction of OGG1 activity is not a result of induction of OGG1 gene expression as assessed by qPCR suggesting a role for protein stabilisation or increased OGG1 catalytic activity. High resolution confocal microscopy pinpointed the location of the fluorescent molecular beacon in live cells to perinuclear regions that were identified as mitochondria by co-staining with mitotracker dye. There is no evidence of cut beacon within the nuclear compartment of the cell. Control experiments with a positive control beacon (G:C base pair and lacking the DAB quencher) did not result in mitochondrial localisation of fluorescence signal indicating that the dye does not accumulate in mitochondria independent of OGG1 activity. Furthermore, faint nuclear staining was apparent confirming that the beacon structure is able to enter the nucleus. In conclusion, these data indicate that the mitochondria are the major site for OGG1 repair activity under conditions of oxidative stress.
Mirbahai
Leda
L
School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
Kershaw
Rachael M
RM
Green
Richard M
RM
Hayden
Rachel E
RE
Meldrum
Rosalind A
RA
Hodges
Nikolas J
NJ
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D001483:Base Sequence; D001959:Bromates / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D045647:DNA Glycosylases / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair / Q000187:drug effects; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D005978:Glutathione / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology; D015335:Molecular Probes / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D018384:Oxidative Stress / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015533:Transcriptional Activation / Q000187:drug effects
D001959:Bromates; D015335:Molecular Probes; C019536:potassium bromate; D000080242:8-Hydroxy-2'-Deoxyguanosine; D045647:DNA Glycosylases; C485990:Ogg1 protein, mouse; D003849:Deoxyguanosine; D005978:Glutathione