The induction of DNA double strand breaks (DSB) may result in chromosomal aberrations or gene mutations, if incorrectly repaired. Identification of a marker of DSB generation or associated repair processes would contribute considerably to the field of mechanistic genotoxicity.
The precise role of the homologous recombination protein RAD52 remains elusive in humans, however the yeast homologue plays a central role in the repair of DSB, and transcriptional up-regulation of human RAD52 has been demonstrated by micro-array analysis and qRT-PCR also. For this reason, we propose to monitor RAD52 transcription by visualising activation of a RAD52 specific molecular beacon using fluorescence microscopy. Previously, we presented data to support the specificity and sensitivity of a RAD52 molecular beacon in a cell free system, in which a significant increase in molecular beacon fluorescence was observed following incubation with complementary oligonucleotide. Molecular beacon design was modified by incorporating a 2’-0 methyl RNA backbone, to diminish non-specific intracellular binding, and here we present evidence for the up-regulation of RAD52 mRNA in whole cells, as indicated by increased molecular beacon fluorescence.
Our recent work has indicated a dose-dependant increase in molecular beacon fluorescence, following exposure of human TK6 lymphoblastoid and A549 lung epithelial cells, to gamma irradiation. Irradiated cells were fixed at 1 or 6h, and samples analysed either for H2AX phosphorylation status, or molecular beacon activation. H2AX phosphorylation is commonly used as a marker of DSBs, however the specificity has recently been questioned, since phosphorylation demonstrates a degree of cell cycle dependence and small foci have been reported in the absence of DNA damaging agents.
Microscopic analysis of irradiated human cells revealed a significant increase in RAD52 molecular beacon activation at 1h, which appears diminished by 6h. Activated molecular beacon appears to aggregate into a number of discrete foci, the frequency of which was elevated by approximately 5-fold and 6-fold after exposure to 0.5 and 1Gy irradiation respectively. In addition to these foci, a homogenous increase of general fluorescence in comparison to unirradiated cells was evident. We have ruled out the possibility of autofluorescence contributing to this effect following irradiation, and by counter-staining with the DNA binding stain DAPI, have located the area of molecular beacon activation to the nucleus. This result provides further evidence for the transcriptional regulation of RAD52 in response to DSB, and to our knowledge is the first result of this kind to be demonstrated in whole cells. In conclusion, we propose our RAD52 mRNA specific molecular beacon as an appropriate method for detecting DSB in whole cells.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA