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Single-molecule studies of the effect of template tension on T7 DNA polymerase activity

Author

Listed:
  • Gijs J.L. Wuite

    (University of California)

  • Steven B. Smith

    (University of California)

  • Mark Young

    (Institute of Molecular Biology, University of Oregon)

  • David Keller

    (University of New Mexico)

  • Carlos Bustamante

    (University of California)

Abstract

T7 DNA polymerase1,2 catalyses DNA replication in vitro at rates of more than 100 bases per second and has a 3′→5′ exonuclease (nucleotide removing) activity at a separate active site. This enzyme possesses a ‘right hand’ shape which is common to most polymerases with fingers, palm and thumb domains3,4. The rate-limiting step for replication is thought to involve a conformational change between an ‘open fingers’ state in which the active site samples nucleotides, and a ‘closed’ state in which nucleotide incorporation occurs3,5. DNA polymerase must function as a molecular motor converting chemical energy into mechanical force as it moves over the template. Here we show, using a single-molecule assay based on the differential elasticity of single-stranded and double-stranded DNA, that mechanical force is generated during the rate-limiting step and that the motor can work against a maximum template tension of ∼34 pN. Estimates of the mechanical and entropic work done by the enzyme show that T7 DNA polymerase organizes two template bases in the polymerization site during each catalytic cycle. We also find a force-induced 100-fold increase in exonucleolysis above 40 pN.

Suggested Citation

  • Gijs J.L. Wuite & Steven B. Smith & Mark Young & David Keller & Carlos Bustamante, 2000. "Single-molecule studies of the effect of template tension on T7 DNA polymerase activity," Nature, Nature, vol. 404(6773), pages 103-106, March.
    • Handle: RePEc:nat:nature:v:404:y:2000:i:6773:d:10.1038_35003614
    DOI: 10.1038/35003614
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    Cited by:

    1. Andreas Walbrun & Tianhe Wang & Michael Matthies & Petr Šulc & Friedrich C. Simmel & Matthias Rief, 2024. "Single-molecule force spectroscopy of toehold-mediated strand displacement," Nature Communications, Nature, vol. 15(1), pages 1-15, December.
    2. Longfu Xu & Matthew T. J. Halma & Gijs J. L. Wuite, 2024. "Mapping fast DNA polymerase exchange during replication," Nature Communications, Nature, vol. 15(1), pages 1-11, December.
    3. Chi, Qingjia & Wang, Guixue & Jiang, Jiahuan, 2013. "The persistence length and length per base of single-stranded DNA obtained from fluorescence correlation spectroscopy measurements using mean field theory," Physica A: Statistical Mechanics and its Applications, Elsevier, vol. 392(5), pages 1072-1079.
    4. Jack W. Shepherd & Sebastien Guilbaud & Zhaokun Zhou & Jamieson A. L. Howard & Matthew Burman & Charley Schaefer & Adam Kerrigan & Clare Steele-King & Agnes Noy & Mark C. Leake, 2024. "Correlating fluorescence microscopy, optical and magnetic tweezers to study single chiral biopolymers such as DNA," Nature Communications, Nature, vol. 15(1), pages 1-15, December.
    5. Ehsan Akbari & Melika Shahhosseini & Ariel Robbins & Michael G. Poirier & Jonathan W. Song & Carlos E. Castro, 2022. "Low cost and massively parallel force spectroscopy with fluid loading on a chip," Nature Communications, Nature, vol. 13(1), pages 1-11, December.

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