A molecular beacon approach to detecting RAD52 expression in response to DNA damage in human cells.
Although DNA damage proteins are infrequently regulated at the transcriptional level, RAD52 mRNA levels appear to be significantly induced in human cells following methyl methanesulphonate (MMS) and Etoposide treatment. Studies have so far been limited to biochemical analysis of cellular extracts and we aimed to extend this observation to whole cells. To address this, we have developed a series of molecular beacon (MB) probes that fluoresce upon hybridising with RAD52 mRNA sequence. MB's are synthetic hairpin probes, which generate a significant fluorescent signal only upon hybridising complementary nucleotide. Three MB's are described herein, which display differential sensitivity, specificity and stability. In particular, the suitability of a texas red-labelled DNA MB (TR-MB), a dual-labelled (FAM-TAMRA) fluorescence resonance energy transfer-capable DNA MB (FRET-MB) and a FAM-labelled MB of 2'-O-methylated RNA backbone (FAM-MB) was investigated. We conclude that FAM-MB is most suitable for intracellular applications, and demonstrate a positive correlation between MB fluorescence intensity, RAD52 gene expression and both gamma ionising radiation and MMS concentration in human TK6 cells. RAD52 contribution to DNA repair has been ascribed to its role in homologous recombination (HR) and therefore we propose FAM-MB could be a potential tool for discriminating between substrates of HR and non-homologous end joining (NHEJ).
Riches
Lucy C
LC
Biomolecular Medicine, Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building, London SW7 2AZ, UK.
Lynch
Anthony M
AM
Gooderham
Nigel J
NJ
D018906:Antineoplastic Agents, Alkylating / Q000633:toxicity; D000972:Antineoplastic Agents, Phytogenic / Q000633:toxicity; D001681:Biological Assay; D045744:Cell Line, Tumor; D002474:Cell-Free System; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects*; D004260:DNA Repair; D005047:Etoposide / Q000633:toxicity*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D008741:Methyl Methanesulfonate / Q000633:toxicity*; D051721:Rad52 DNA Repair and Recombination Protein / Q000235:genetics / Q000378:metabolism*
D018906:Antineoplastic Agents, Alkylating; D000972:Antineoplastic Agents, Phytogenic; C497194:RAD52 protein, human; D051721:Rad52 DNA Repair and Recombination Protein; D005047:Etoposide; D008741:Methyl Methanesulfonate