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.

中文翻译：

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电化学装置在表征解旋酶对DNA的动态作用中的应用

关于DNA解旋酶结合和活性的动力学和热力学仍有许多待理解。在这里，我们将探针修饰的DNA单分子层用作多重识别金电极，作为敏感的识别元件和解旋酶与DNA相互作用的形态响应传感器。这些设备发出的电化学信号对解旋酶产生的DNA的结构变形高度敏感。我们使用这种DNA电化学来区分三种不同XPB解旋酶的DNA相互作用的细节，这些XPB解旋酶属于解旋酶的超家族2。解旋酶结合后观察到DNA熔解温度和双链体稳定性的明显变化，这种变化是常规的紫外可见吸收测量所无法观察到的。结合解离常数估计在10至50 nM的范围内，并与活性观察结果相关。还跟踪了ATP刺激的DNA解旋活性，揭示了与常规和分子扳手操作模式相关的指数时间尺度和不同的时间常数，这些进一步的晶体结构进一步证实了这一点。因此，这些设备为解旋酶与DNA相互作用的结构热力学和动力学提供了灵敏的量度。