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  • The EMBO Journal (2009) 28, 2794 - 2802
  • doi:10.1038/emboj.2009.219

Published online: 6 August 2009

Proofreading dynamics of a processive DNA polymerase

Borja Ibarra1,7, Yann R Chemla1,8, Sergey Plyasunov1,9, Steven B Smith1, José M Lázaro2, Margarita Salas2 and Carlos Bustamante1,3,4,5,6

  1. Department of Physics, University of California, Berkeley, CA, USA
  2. Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Madrid, Spain
  3. Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA
  4. Department of Chemistry, University of California, Berkeley, CA, USA
  5. Howard Hughes Medical Institute, University of California, Berkeley, CA, USA
  6. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

Correspondence to:

Carlos Bustamante, Department of Physics, QB3, Howard Hughes Medical Institute, University of California Berkeley, 608 Stanley Hall, Berkeley, CA 94720-3220, USA. Tel.: +510 643 9706; Fax: +510 643-4500; E-mail: carlos@alice.berkeley.edu

7Present address: Departamento Estructura de Macromoléculas, Centro Nacional de Biotecnología (CSIC), Madrid, Madrid 28049, Spain

8Present address: Department of Physics, University of Illinois, Urbana, IL 61801, USA

9Present address: SIG LLP. Bala Cynwyd, PA 19004, USA

Received 28 April 2009; Accepted 6 July 2009

Replicative DNA polymerases present an intrinsic proofreading activity during which the DNA primer chain is transferred between the polymerization and exonuclease sites of the protein. The dynamics of this primer transfer reaction during active polymerization remain poorly understood. Here we describe a single-molecule mechanical method to investigate the conformational dynamics of the intramolecular DNA primer transfer during the processive replicative activity of the Phi29 DNA polymerase and two of its mutants. We find that mechanical tension applied to a single polymerase–DNA complex promotes the intramolecular transfer of the primer in a similar way to the incorporation of a mismatched nucleotide. The primer transfer is achieved through two novel intermediates, one a tension-sensitive and functional polymerization conformation and a second non-active state that may work as a fidelity check point for the proofreading reaction.

  • Keywords:

    • bacteriophage Phi29,
    • DNA replication,
    • molecular motors,
    • optical tweezers,
    • proofreading