Here we used Nanopore Tweezers (SPRNT) to examine the behavior of a model helicase PcrA while walking along single-stranded DNA and unwinding double-stranded DNA. Nanopore tweezers allows for the application of assisting and opposing forces. We used that here to examine how PcrA responds to such forces. Our results reveal new details about how PcrA walks and unwinds DNA. Surprisingly, PcrA is highly sequence dependent in both single-stranded DNA translocation and double-stranded DNA unwinding, and the sequence dependence of translocation has a significant effect on how readily double-stranded DNA can be unwound. This is in stark contrast to the conventional wisdom that the DNA bases being unwound should dominate the behavior as they make up the primary work of unwinding DNA. While the DNA duplex does affect the rate of unwinding, our data reveal that PcrA's ability to walk along the underlying single-stranded DNA also plays an important role.
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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).