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Application of Electrochemical Devices to Characterize the Dynamic Actions of Helicases on DNA
journal contribution
posted on 2017-12-29, 00:00 authored by Dimithree Kahanda, Kevin T. DuPrez, Eduardo Hilario, Marc A. McWilliams, Chris H. Wohlgamuth, Li Fan, Jason D. SlinkerMuch 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
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