Proofreading dynamics of a processive DNA polymerase

Borja Ibarra, Yann R. Chemla, Sergey Plyasunov, Steven B. Smith, José M. Lázaro, Margarita Salas, Carlos Bustamante

Research output: Contribution to journalArticle

Abstract

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 29 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.

Original languageEnglish (US)
Pages (from-to)2794-2802
Number of pages9
JournalEMBO Journal
Volume28
Issue number18
DOIs
StatePublished - Sep 16 2009

Keywords

  • Bacteriophage Phi29
  • DNA replication
  • Molecular motors
  • Optical tweezers
  • Proofreading

ASJC Scopus subject areas

  • Neuroscience(all)
  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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    Ibarra, B., Chemla, Y. R., Plyasunov, S., Smith, S. B., Lázaro, J. M., Salas, M., & Bustamante, C. (2009). Proofreading dynamics of a processive DNA polymerase. EMBO Journal, 28(18), 2794-2802. https://doi.org/10.1038/emboj.2009.219