Single-molecule studies of the effect of template tension on T7 DNA polymerase activity (original) (raw)

Nature volume 404, pages 103–106 (2000)Cite this article

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.

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Acknowledgements

We thank J. Davenport and M. Hegner for their help with the template and suggestions, and D. Bensimon, B. Maier and V. Croquette for sharing their results before publication.

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Authors and Affiliations

  1. Department of Physics and Department of Molecular and Cell Biology, University of California, Berkeley , 94720, California, USA
    Gijs J.L. Wuite, Steven B. Smith & Carlos Bustamante
  2. Institute of Molecular Biology, University of Oregon, Eugene, 97403, Oregon, USA
    Mark Young
  3. Department of Chemistry, University of New Mexico, Albuquerque, 87131 , New Mexico, USA
    David Keller

Authors

  1. Gijs J.L. Wuite
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  2. Steven B. Smith
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  3. Mark Young
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  4. David Keller
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  5. Carlos Bustamante
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Corresponding author

Correspondence toCarlos Bustamante.

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Wuite, G., Smith, S., Young, M. et al. Single-molecule studies of the effect of template tension on T7 DNA polymerase activity.Nature 404, 103–106 (2000). https://doi.org/10.1038/35003614

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