A molecular beacon assay for measuring base excision repair activities Andrei Maksimenko, a Alexander A. Ishchenko, b,1 Guenha€ el Sanz, b,2 Jacques Laval, b Rhoderick H. Elder, c and Murat K. Saparbaev b, * a BioAlliance Pharma SA, 59, Bvd du G en eral Martial Valin, 75015 Paris, France b Groupe “R eparation de l’ADN,” UMR 8113 CNRS, LBPA-ENS Cachan, Institut Gustave Roussy, 39, rue Camille Desmoulins, 94805 Villejuif Cedex, France c Cancer Research UK Carcinogenesis Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester, M20 4BX, UK Received 17 March 2004 Available online 14 May 2004 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 de- signed a single-stranded DNA oligonucleotide labelled with a 5 0 -fluorescein (F) and a 3 0 -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. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Molecular beacon; DNA glycosylase; AP endonuclease; Base excision repair; Nucleotide incision repair; Oxidative DNA damage Reactive oxygen species-induced non-bulky DNA damage is believed to contribute significantly to cell le- thality, tissue degeneration, ageing, and cancer [1]. To counteract the deleterious effects of these lesions, which may lead to genomic instability, cells have evolved a number of DNA repair mechanisms, including the base excision repair (BER) pathway [2]. In BER, DNA gly- cosylases recognize and remove damaged and/or mi- spaired bases from DNA by cleavage of the N-glycosylic bond between the abnormal base and deoxyribose, leav- ing either an abasic site or a single-strand nick in DNA [3]. As the biological consequences of persisting endoge- nously and exogenously generated DNA modifications, together with variations in DNA repair activities are connected to both individual and population disease susceptibility [4,5], the development of rapid and high- throughput screening assays for BER activities is of fundamental importance. Current assays for DNA gly- cosylase and AP endonuclease activities are time-con- suming and indirect since they are based on separation techniques such as solubility in acid and/or ethanol [6], chromatography [7], gel electrophoresis [8], paramagnetic beads [9], etc. Generally, DNA substrates are radioac- tively labelled to measure BER activity in a quantitative manner, although non-radioactive, fluorometric oligo- nucleotide assays coupled to gel electrophoresis have also been described [10]. Importantly, the requirement for an additional step to separate the reaction product precludes * Corresponding author. Fax: +331-42115276. E-mail address: smurat@igr.fr (M.K. Saparbaev). 1 Permanent address: Novosibirsk Institute of Chemical Biology and Fundamental Medicine, Siberian Division of the Russian Acad- emy of Sciences, Novosibirsk 630090, Russia. 2 Present address: Unit e de Recherches de Biochimie et Structure des Prot eines, INRA, Domaine de Vilvert, B^ atiment 526, 78352 Jouy- en-Josas Cedex, France. 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.04.179 Biochemical and Biophysical Research Communications 319 (2004) 240–246 BBRC www.elsevier.com/locate/ybbrc