A molecular beacon assay for measuring base excision repair activities.
The base excision repair (BER) pathway plays a key role in protecting the genome from endogenous DNA damage. Current methods to measure BER activities are indirect and cumbersome. Here, we introduce a direct method to assay DNA excision repair that is suitable for automation and industrial use, based on the fluorescence quenching mechanism of molecular beacons. We designed a single-stranded DNA oligonucleotide labelled with a 5'-fluorescein (F) and a 3'-Dabcyl (D) in which the fluorophore, F, is held in close proximity to the quencher, D, by the stem-loop structure design of the oligonucleotide. Following removal of the modified base or incision of the oligonucleotide, the fluorophore is separated from the quencher and fluorescence can be detected as a function of time. Several modified beacons have been used to validate the assay on both cell-free extracts and purified proteins. We have further developed the method to analyze BER in cultured cells. As described, the molecular beacon-based assay can be applied to all DNA modifications processed by DNA excision/incision repair pathways. Possible applications of the assay are discussed, including high-throughput real-time DNA repair measurements both in vitro and in living cells.
Maksimenko
Andrei
A
BioAlliance Pharma SA, 59, Bvd du Général Martial Valin, 75015 Paris, France.
Ishchenko
Alexander A
AA
Sanz
Guenhaël
G
Laval
Jacques
J
Elder
Rhoderick H
RH
Saparbaev
Murat K
MK
D000818:Animals; D002474:Cell-Free System; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D045647:DNA Glycosylases / Q000737:chemistry; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism; D005821:Genetic Techniques; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry; D010100:Oxygen / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D009841:Oligonucleotides; D004247:DNA; D045647:DNA Glycosylases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D010100:Oxygen