title: A fluorescence-based helicase assay: application to the screening of G-quadruplex ligands.
abstract: Helicases, enzymes that unwind DNA or RNA structure, are present in the cell nucleus and in the mitochondrion. Although the majority of the helicases unwind DNA or RNA duplexes, some of these proteins are known to resolve unusual structures such as G-quadruplexes (G4) in vitro. G4 may form stable barrier to the progression of molecular motors tracking on DNA. Monitoring G4 unwinding by these enzymes may reveal the mechanisms of the enzymes and provides information about the stability of these structures. In the experiments presented herein, we developed a reliable, inexpensive and rapid fluorescence-based technique to monitor the activity of G4 helicases in real time in a 96-well plate format. This system was used to screen a series of G4 structures and G4 binders for their effect on the Pif1 enzyme, a 5' to 3' DNA helicase. This simple assay should be adaptable to analysis of other helicases and G4 structures.
authors.affiliation: University of Bordeaux, ARNA laboratory, Bordeaux, France INSERM, U869, IECB, Pessac, France oscar.mendoza@u-bordeaux.fr.
authors.forename: Oscar
authors.initials: O
authors.lastname: Mendoza
authors.affiliation: University of Bordeaux, ARNA laboratory, Bordeaux, France INSERM, U869, IECB, Pessac, France.
authors.forename: Nassima Meriem
authors.initials: NM
authors.lastname: Gueddouda
authors.affiliation: CNRS, UMR7196, Muséum National d'Histoire Naturelle, 75005 Paris, France.
authors.forename: Jean-Baptiste
authors.initials: JB
authors.lastname: Boulé
authors.affiliation: INSERM, U869, IECB, Pessac, France Univ. Poitiers, 40 avenue du recteur Pineau, 86000 Poitiers, France a.bourdoncle@u-bordeaux.fr.
authors.forename: Anne
authors.initials: A
authors.lastname: Bourdoncle
authors.affiliation: University of Bordeaux, ARNA laboratory, Bordeaux, France INSERM, U869, IECB, Pessac, France.
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Mergny
mesh_terms: D004265:DNA Helicases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005453:Fluorescence; D054856:G-Quadruplexes; D008024:Ligands
chemical_list: D004791:Enzyme Inhibitors; D008024:Ligands; D004265:DNA Helicases