A fluorescence-based helicase assay: application to the screening of G-quadruplex ligands

Nucleic Acids Research, Jun 2015

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.

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A fluorescence-based helicase assay: application to the screening of G-quadruplex ligands

Nucleic Acids Research A fluorescence-based helicase assay: application to the screening of G-quadruplex ligands Oscar Mendoza 1 2 Nassima Meriem Gueddouda 1 2 Jean-Baptiste Boul e´ 0 Anne Bourdoncle 1 3 Jean-Louis Mergny 1 2 0 CNRS, UMR7196, Mus e ́um National d'Histoire Naturelle , 75005 Paris , France 1 INSERM, U869, IECB , Pessac , France 2 University of Bordeaux, ARNA laboratory , Bordeaux , France 3 Univ. Poitiers , 40 avenue du recteur Pineau, 86000 Poitiers , France 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 Gquadruplexes (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 fluorescencebased 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. - Helicases are a central class of enzymes found in all known organisms. By a combination of biochemical and genetic approaches, their activity has been shown to affect most metabolic processes that rely on nucleic acid unwinding in the cell, including DNA replication, transcription, translation, DNA repair or recombination. At the molecular level, helicases are motor proteins that move directionally along a nucleic acid phosphodiester backbone and are able to separate annealed nucleic acid strands using energy derived from adenosine triphosphate (ATP) hydrolysis. Although the majority of the known helicase enzymes are involved in the process of unwinding duplex DNA or RNA, some of these proteins have been known to act on less canonical substrates like protein-nucleic acids complexes or secondary DNA structures, in particular G-quadruplexes (G4) (1–4). In the G4 field, helicase studies have been at the forefront for demonstrating the formation of these structures in vivo, through various biochemical and genetic approaches (5–10). Some of the outstanding questions in the field are now how specific helicases are to various Gquadruplex structures, or what is the degree of redundancy between different helicases in their capacity to process these structures. It is well documented that G4 can adopt multiple structures and strand arrangements, which differ thermodynamically (11). How these affect the helicase activity is currently poorly assessed. Owing to technical limitations and to the relative youth of this field, G-quadruplex unwinding has been monitored for a limited number of helicases, including members of the RecQ family (12,13), the XPD family (FANCJ) (3) and the Pif1 family (2,14,15) and for a limited number of substrates. The most common assay for measuring helicase activity in vitro employs gel electrophoresis (16). Electrophoresis has the obvious advantage to allow the determination of the diversity and relative abundance of the molecular species present in the reaction. This is however a relatively cumbersome and low-throughput technique (17,18). Several additional methods based on fluorescence assays have been developed to overcome some of the limitations of electrophoresis, and to tackle real-time kinetics aspects of helicase action (3,19). To improve tools aiming at characterizing the activity of DNA helicases towards G4 structures, we developed a fluorescence-based helicase assay that allows testing the unwinding of different G4 structures by a helicase in high throughput and in real time. In this work, we demonstrate its application to study the activity of the DNA helicase Pif1p, a prototypal member of the PIF1 family of DNA helicase, and for which substantial evidences have been accumulated suggesting its activity on G4 in vitro and in vivo (15,19,20). This assay is also used to assess the inhibitory effect of pharmacological G4 binders on the activity of Pif1p. To our knowledge this is the first real-time helicase assay developed that allows, in a single experiment, the screening of series G4 sequences under several conditions and in the presence of G4 ligands. Moreover, this assay should be adaptable to the specific requirements of other helicase enzymes. MATERIALS AND METHODS Oligonucleotides and compounds All oligonucleotides used in this research were purchased from Eurogentec and stored at −20◦C as 100–200 M stock solutions. Oligonucleotide strand concentrations were determined by absorbance at 260 nm using the extinction coefficients provided by the manufacturer. Sequences are provided in Table 1. DNA sy (...truncated)


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Oscar Mendoza, Nassima Meriem Gueddouda, Jean-Baptiste Boulé, Anne Bourdoncle, Jean-Louis Mergny. A fluorescence-based helicase assay: application to the screening of G-quadruplex ligands, Nucleic Acids Research, 2015, pp. e71-e71, 43/11, DOI: 10.1093/nar/gkv193