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Proofreading dynamics of a processive DNA polymerase |
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| Authors: | Steven B Smith José M Lázaro Margarita Salas Carlos Bustamante |
| Affiliation: | 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 |
| 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 Φ29 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 |