DNA helicases use the energy of ATP-hydrolysis to walk along and unwind DNA, making them essential for many cellular tasks. Still, key details of how they work are not well understood because their small (0.6 nanometer) and fast (~1 millisecond) steps make them difficult to study using traditional biophysical approaches. Here we apply Nanopore Tweezers, which is capable of clearly resolving these steps, to shed light on how a prototypical helicase called PcrA works. By changing different aspects of the Nanopore Tweezers experiment, we probe different substates of the ATPase cycle. Nanopore Tweezers provides an enzyme’s sequence-specific location and surprisingly reveals that the underlying single-stranded DNA sequence strongly affects the ability of PcrA to unwind double stranded DNA.
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Sequence-dependent mechanochemical coupling of helicase translocation and unwinding at single-nucleotide resolution.
A. H. Laszlo, J. M. Craig, M. Gavrilov, R. Tippana, I. C. Nova, J. R. Huang, H. C. Kim, S. J. Abell, M. deCampos-Stairiker, J. W. Mount, J. L. Bowman, K. S. Baker, H. Higinbotham, D. Bobrovnikov, T. Ha, J. H. Gundlach, Sequence-dependent mechanochemical coupling of helicase translocation and unwinding at single-nucleotide resolution. Proc. Natl. Acad. Sci. 119, e2202489119 (2022).