An oligonucleotide microarray for the monitoring of repair enzyme activity toward different DNA base damage.
Characterization of DNA-N-glycosylase activities in cell extract is a challenging problem and could represent a major concern for medical applications. Synthetic oligonucleotides which contain base lesions located on specific sites constitute suitable substrates for their study. An in vitro miniaturized assay was developed that allows the measurement of cleavage activities of DNA repair enzymes on a set of oligonucleotides (ODNs) that contained different lesions. The modified ODNs were indirectly hybridized onto probes chemically fixed at defined sites on a circular format within each well of a 96-well microtiter plate (Oligo Sorbent Array, OLISA). The lesions were selected among oxidative damage (8-oxo-7,8-dihydroguanine, formylamine), deaminated bases (uracil, hypoxanthine) and alkylated base (N(6)-etheno-adenine). Cleavage specificity was checked using different enzymes: Fapy-DNA-N-glycosylase, 3-methyladenine DNA glycosylase II, uracil-N-glycosylase, endonuclease V and endonuclease VIII. The extent of excision could be monitored simultaneously for the selected base damage. For this purpose, we used automated fluorescence imaging analysis of the residual ODNs that contained lesions and remained on the support after release of the cleaved ODNs recognized by the repair enzymes. The results indicated that this assay could advantageously replace the analysis of glycosylase activities by PAGE techniques. Finally we show that this in vitro repair assay represents an interesting tool for the determination of cellular repair activities.
Sauvaigo
Sylvie
S
Laboratoire des Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, CEA/DSM/Département de Recherche Fondamentale sur la Matiére Condensée, CEA-Grenoble, 17 rue des martyrs, 38054 Grenoble Cedex 9, France. ssauvaigo@cea.fr
Guerniou
Valérie
V
Rapin
Delphine
D
Gasparutto
Didier
D
Caillat
Sylvain
S
Favier
Alain
A
D004249:DNA Damage; D045647:DNA Glycosylases / Q000032:analysis*; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000032:analysis*; D005347:Fibroblasts / Q000201:enzymology; D006367:HeLa Cells; D006801:Humans; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry*
D009841:Oligonucleotides; D043224:Deoxyribonuclease (Pyrimidine Dimer); D045647:DNA Glycosylases