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Proofreading dynamics of a processive DNA polymerase

AuthorsIbarra, Borja; Chemla, Y. R.; Plyasunov, Sergey; Smith, Steven B.; Lázaro, José M. ; Salas, Margarita ; Bustamante, C.
KeywordsBacteriophage Phi29
DNA replication
Molecular motors
Optical tweezers
Issue Date6-Aug-2009
PublisherEuropean Molecular Biology Organization
Nature Publishing Group
CitationEmbo Journal 25, 1335-1343 (2006)
AbstractReplicative 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.
Publisher version (URL)http://dx.doi.org/10.1038/emboj.2009.219
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