Electronic measurements of single-molecule processing by DNA polymerase I

ORAL

Abstract

A single-molecule nanocircuit technique is applied to continuously monitor DNA replication activity by the enzyme DNA polymerase I (Pol I). Using single copies of Pol I bound to a single-walled carbon nanotube device, an electrical signal was generated to reveal enzymatic function and dynamic variability. Continuous, single-molecule-resolution recordings were obtained for Pol I processing homopolymeric DNA templates over 10 minutes and through \textgreater 10,000 DNA replication events. Processivity of up to 40 nucleotide bases was directly observed, and statistical analysis of the recordings determined key kinetic parameters for the enzyme's open and closed conformations. We observe that the closed complex forms a phosphodiester bond in a highly efficient process \textgreater 99.8{\%} of the time, with a mean duration of only 0.3 ms for all four dNTPs. The rate-limiting step for replication occurs during the enzyme's open state, but with a duration that is nearly twice as long for dATP or dTTP incorporation than for dCTP or dGTP. Taken together, the results provide a wealth of new information complementing prior work on the mechanism and dynamics of DNA polymerase.

–

Authors

  • Yongki Choi

    • Department of Physics and Astronomy, University of California, Irvine
    • Departments of Physics and Astronomy, Molecular Biology and Biochemistry, and Chemistry, University of California Irvine, Irvine, CA 92697

  • Tivoli Olsen

    • Department of Chemistry, University of California, Irvine

  • Tolga Gul

    • Department of Physics and Astronomy, University of California, Irvine

  • Brad L. Corso

    • Department of Physics and Astronomy, University of California, Irvine
    • University of California Irvine

  • Chengjun Dong

    • Department of Physics and Astronomy, University of California, Irvine

  • William Brown

    • Department of Chemistry, University of California, Irvine

  • Gregory A. Weiss

    • Department of Molecular Biology, University of California, Irvine
    • Departments of Molecular Biology and Biochemistry, and Chemistry, University of California, Irvine

  • Phillip Collins

    • Department of Physics and Astronomy, University of California, Irvine
    • University of California, Irvine
    • University of California Irvine
    • University of California, Riverside