A new kinetic assay that monitors helicase unwinding of G-quadruplexes

Authors

• HS Shamroukh
• MT Banco
• AR Ferre-D'Amare

Abstract

G-quadruplexes (G4) are four-stranded nucleic acid structures formed by guanine-rich sequences that fold into stacked G-quartets. G4s regulate numerous cellular processes wherein helicases are required to catalyze unwinding of the highly stable structure. Several helicases exhibit G4-unwinding activity, but the molecular basis of G4 selectivity, binding and unwinding are still under active study. Although several approaches to biochemical characterization of helicases exist, quantitation of helicase unwinding of intramolecular G4s, the predominant form found in cells, is challenging due to the high propensity for the helicase-destabilized nucleic acids to refold upon release from the enzymes. To overcome this, most helicase assays require the inclusion of a large excess (100 to 1000-fold) of trap strand. We have now developed a new fluorescence-based kinetic assay that measures the G4-unwinding activity of helicases using substrates containing a bimolecular G4 structure. Our approach overcomes the requirement of a trap strand due to the slow intrinsic refolding rate of bimolecular G4s. To demonstrate the utility of this assay, we analyzed the unwinding kinetics of the major G4 helicase from vertebrates, DHX36, in steady-state and single turnover conditions with various substrates. Altogether, this method presents a simple and quantitative approach to characterizing the unwinding of G4s by helicases. We are currently extending is application to high-throughput screening of small-molecules libraries against these essential motor proteins.

Scientific Focus Area: RNA Biology

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