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Proofreading dynamics of a processive DNA polymerase.

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 Phi 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.

SUBMITTER: Ibarra B

PROVIDER: S-EPMC2750014 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Proofreading dynamics of a processive DNA polymerase.

Ibarra Borja B Chemla Yann R YR Plyasunov Sergey S Smith Steven B SB Lázaro José M JM Salas Margarita M Bustamante Carlos C

The EMBO journal 20090806 18

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 Phi 29 DNA polymerase and ...[more]

PMID: 19661923

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