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Abstract
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.
Copyright 2009 Elsevier B.V. All rights reserved.