Application of Electrochemical Devices to Characterize the Dynamic Actions of Helicases on DNA
journal contributionposted on 29.12.2017, 00:00 by Dimithree Kahanda, Kevin T. DuPrez, Eduardo Hilario, Marc A. McWilliams, Chris H. Wohlgamuth, Li Fan, Jason D. Slinker
Much remains to be understood about the kinetics and thermodynamics of DNA helicase binding and activity. Here, we utilize probe-modified DNA monolayers on multiplexed gold electrodes as a sensitive recognition element and morphologically responsive transducer of helicase–DNA interactions. The electrochemical signals from these devices are highly sensitive to structural distortion of the DNA produced by the helicases. We used this DNA electrochemistry to distinguish the details of the DNA interactions of three distinct XPB helicases, which belong to the superfamily-2 of helicases. Clear changes in DNA melting temperature and duplex stability were observed upon helicase binding, shifts that could not be observed with conventional UV–visible absorption measurements. Binding dissociation constants were estimated in the range from 10 to 50 nM and correlated with observations of activity. ATP-stimulated DNA unwinding activity was also followed, revealing exponential time scales and distinct time constants associated with conventional and molecular wrench modes of operation further confirmed by crystal structures. These devices thus provide a sensitive measure of the structural thermodynamics and kinetics of helicase–DNA interactions.
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time scalesClear changesXPB helicasestime constantsduplex stabilityDNA helicase bindingmultiplexed gold electrodesDNA electrochemistry50 nMDNA interactionsElectrochemical Devicescrystal structuressuperfamily -2Binding dissociation constantsprobe-modified DNA monolayersATP-stimulated DNA unwinding activityhelicase bindingelectrochemical signalsUVDynamic Actionsrecognition element