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Dangerfield TL, Kirmizialtin S, Johnson KA. Conformational dynamics during misincorporation and mismatch extension defined using a DNA polymerase with a fluorescent artificial amino acid. J Biol Chem. 2021;298(1):101451doi: 10.1016/j.jbc.2021.101451.
Dangerfield, T. L., Kirmizialtin, S., & Johnson, K. A. (2021). Conformational dynamics during misincorporation and mismatch extension defined using a DNA polymerase with a fluorescent artificial amino acid. The Journal of biological chemistry, 298(1), 101451. https://doi.org/10.1016/j.jbc.2021.101451
Dangerfield, Tyler L, et al. "Conformational dynamics during misincorporation and mismatch extension defined using a DNA polymerase with a fluorescent artificial amino acid." The Journal of biological chemistry vol. 298,1 (2021): 101451. doi: https://doi.org/10.1016/j.jbc.2021.101451
Dangerfield TL, Kirmizialtin S, Johnson KA. Conformational dynamics during misincorporation and mismatch extension defined using a DNA polymerase with a fluorescent artificial amino acid. J Biol Chem. 2021 Nov 25;298(1):101451. doi: 10.1016/j.jbc.2021.101451. Epub 2021 Nov 25. PMID: 34838820; PMCID: PMC8715121.
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J Biol Chem. 2021 Nov 25;298(1):101451. doi: 10.1016/j.jbc.2021.101451. Epub 2021 Nov 25.

Conformational dynamics during misincorporation and mismatch extension defined using a DNA polymerase with a fluorescent artificial amino acid.

The Journal of biological chemistry

Tyler L Dangerfield, Serdal Kirmizialtin, Kenneth A Johnson

Affiliations

  1. Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas, USA.
  2. Chemistry Program, Division of Science, New York University Abu Dhabi, Abu Dhabi, United Arab Emirates.
  3. Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas, USA. Electronic address: [email protected].

PMID: 34838820 PMCID: PMC8715121 DOI: 10.1016/j.jbc.2021.101451

Abstract

High-fidelity DNA polymerases select the correct nucleotide over the structurally similar incorrect nucleotides with extremely high specificity while maintaining fast rates of incorporation. Previous analysis revealed the conformational dynamics and complete kinetic pathway governing correct nucleotide incorporation using a high-fidelity DNA polymerase variant containing a fluorescent unnatural amino acid. Here we extend this analysis to investigate the kinetics of nucleotide misincorporation and mismatch extension. We report the specificity constants for all possible misincorporations and characterize the conformational dynamics of the enzyme during misincorporation and mismatch extension. We present free energy profiles based on the kinetic measurements and discuss the effect of different steps on specificity. During mismatch incorporation and subsequent extension with the correct nucleotide, the rates of the conformational change and chemistry are both greatly reduced. The nucleotide dissociation rate, however, increases to exceed the rate of chemistry. To investigate the structural basis for discrimination against mismatched nucleotides, we performed all atom molecular dynamics simulations on complexes with either the correct or mismatched nucleotide bound at the polymerase active site. The simulations suggest that the closed form of the enzyme with a mismatch bound is greatly destabilized due to weaker interactions with active site residues, nonideal base pairing, and a large increase in the distance from the 3'-OH group of the primer strand to the α-phosphate of the incoming nucleotide, explaining the reduced rates of misincorporation. The observed kinetic and structural mechanisms governing nucleotide misincorporation reveal the general principles likely applicable to other high-fidelity DNA polymerases.

Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.

Keywords: DNA polymerase; conformational change; enzyme kinetics; fluorescence; misincorporation; mismatch; protein dynamics; unnatural amino acid

Conflict of interest statement

Conflict of interest K. A. J. is president of KinTek Corporation, which provided the SF-300x stopped-flow instrument and KinTek Explorer software used in this study.

Publication Types

Grant support