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A molecular beacon assay for measuring base excision repair activities.
Maksimenko, Andrei Ishchenko, Alexander A Sanz, Guenhaƫl Laval, Jacques Elder, Rhoderick H Saparbaev, Murat K
Maksimenko, Andrei
Ishchenko, Alexander A
Sanz, Guenhaƫl
Laval, Jacques
Elder, Rhoderick H
Saparbaev, Murat K
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Abstract
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.
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Date
2004-06-18
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Article
Citation
A molecular beacon assay for measuring base excision repair activities. 2004, 319 (1):240-6 Biochem. Biophys. Res. Commun.