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Reduced structural flexibility for an exonuclease deficient DNA polymerase III mutant

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Title: Reduced structural flexibility for an exonuclease deficient DNA polymerase III mutant
Authors: Gahlon, HL
Walker, AR
Cisneros, GA
Lamers, MH
Rueda, DS
Item Type: Journal Article
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.
Issue Date: 22-Oct-2018
Date of Acceptance: 3-Sep-2018
URI: http://hdl.handle.net/10044/1/64099
DOI: https://dx.doi.org/10.1039/C8CP04112A
ISSN: 1463-9076
Publisher: Royal Society of Chemistry
Start Page: 26892
End Page: 26902
Journal / Book Title: Physical Chemistry Chemical Physics
Volume: 20
Issue: 42
Copyright Statement: © 2018 the Owner Societies. This Open Access Article is licensed under a Creative Commons Attribution 3.0 Unported Licence https://creativecommons.org/licenses/by/3.0/
Sponsor/Funder: Commission of the European Communities
Marie Skłodowska Curie Actions
Medical Research Council
Funder's Grant Number: 705015
705015
MC_UP_1102/5
Keywords: 02 Physical Sciences
03 Chemical Sciences
Chemical Physics
Publication Status: Published
Appears in Collections:Department of Medicine
Faculty of Medicine



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