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Issue 42, 2018
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Reduced structural flexibility for an exonuclease deficient DNA polymerase III mutant

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Abstract

DNA synthesis, carried out by DNA polymerases, requires balancing speed and accuracy for faithful replication of the genome. High fidelity DNA polymerases contain a 3′–5′ exonuclease domain that can remove misincorporated nucleotides on the 3′ end of the primer strand, a process called proofreading. The E. coli replicative polymerase, DNA polymerase III, has spatially separated (∼55 Å apart) polymerase and exonuclease subunits. Here, we report on the dynamics of E. coli DNA polymerase III proofreading in the presence of its processivity factor, the β2-sliding clamp, at varying base pair termini using single-molecule FRET. We find that the binding kinetics do not depend on the base identity at the termini, indicating a tolerance for DNA mismatches. Further, our single-molecule data and MD simulations show two previously unobserved features: (1) DNA Polymerase III is a highly dynamic protein that adopts multiple conformational states while bound to DNA with matched or mismatched ends, and (2) an exonuclease-deficient DNA polymerase III has reduced conformational flexibility. Overall, our single-molecule experiments provide high time-resolution insight into a mechanism that ensures high fidelity DNA replication to maintain genome integrity.

Graphical abstract: Reduced structural flexibility for an exonuclease deficient DNA polymerase III mutant

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Publication details

The article was received on 29 Jun 2018, accepted on 03 Sep 2018 and first published on 22 Oct 2018


Article type: Paper
DOI: 10.1039/C8CP04112A
Citation: Phys. Chem. Chem. Phys., 2018,20, 26892-26902
  • Open access: Creative Commons BY license
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    Reduced structural flexibility for an exonuclease deficient DNA polymerase III mutant

    H. L. Gahlon, A. R. Walker, G. A. Cisneros, M. H. Lamers and D. S. Rueda, Phys. Chem. Chem. Phys., 2018, 20, 26892
    DOI: 10.1039/C8CP04112A

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