Journal Article

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

Oscar Mendoza, Nassima Meriem Gueddouda, Jean-Baptiste Boulé, Anne Bourdoncle and Jean-Louis Mergny

in Nucleic Acids Research

Volume 43, issue 11, pages e71-e71
Published in print June 2015 | ISSN: 0305-1048
Published online March 2015 | e-ISSN: 1362-4962 | DOI: http://dx.doi.org/10.1093/nar/gkv193

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  • Research Methods in Life Sciences
  • Enzymology
  • Molecular Biology and Genetics
  • Proteins

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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.

Journal Article.  6241 words.  Illustrated.

Subjects: Research Methods in Life Sciences ; Enzymology ; Molecular Biology and Genetics ; Proteins

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