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Title: The Structure of a High Fidelity DNA Polymerase Bound to a Mismatched Nucleotide Reveals an ;Ajar; Intermediate Conformation in the Nucleotide Selection Mechanism

Abstract

To achieve accurate DNA synthesis, DNA polymerases must rapidly sample and discriminate against incorrect nucleotides. Here we report the crystal structure of a high fidelity DNA polymerase I bound to DNA primer-template caught in the act of binding a mismatched (dG:dTTP) nucleoside triphosphate. The polymerase adopts a conformation in between the previously established 'open' and 'closed' states. In this 'ajar' conformation, the template base has moved into the insertion site but misaligns an incorrect nucleotide relative to the primer terminus. The displacement of a conserved active site tyrosine in the insertion site by the template base is accommodated by a distinctive kink in the polymerase O helix, resulting in a partially open ternary complex. We suggest that the ajar conformation allows the template to probe incoming nucleotides for complementarity before closure of the enzyme around the substrate. Based on solution fluorescence, kinetics, and crystallographic analyses of wild-type and mutant polymerases reported here, we present a three-state reaction pathway in which nucleotides either pass through this intermediate conformation to the closed conformation and catalysis or are misaligned within the intermediate, leading to destabilization of the closed conformation.

Authors:
;  [1]
  1. (Duke)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Institutes of Health (NIH)
OSTI Identifier:
1021781
Resource Type:
Journal Article
Resource Relation:
Journal Name: J. Biol. Chem.; Journal Volume: 286; Journal Issue: (22) ; 06, 2011
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; CATALYSIS; CLOSURES; CRYSTAL STRUCTURE; CRYSTALLOGRAPHY; DNA; DNA POLYMERASES; ENZYMES; FLUORESCENCE; KINETICS; MUTANTS; NUCLEOSIDES; NUCLEOTIDES; POLYMERASES; PROBES; SYNTHESIS; TYROSINE

Citation Formats

Wu, Eugene Y., and Beese, Lorena S. The Structure of a High Fidelity DNA Polymerase Bound to a Mismatched Nucleotide Reveals an ;Ajar; Intermediate Conformation in the Nucleotide Selection Mechanism. United States: N. p., 2011. Web. doi:10.1074/jbc.M110.191130.
Wu, Eugene Y., & Beese, Lorena S. The Structure of a High Fidelity DNA Polymerase Bound to a Mismatched Nucleotide Reveals an ;Ajar; Intermediate Conformation in the Nucleotide Selection Mechanism. United States. doi:10.1074/jbc.M110.191130.
Wu, Eugene Y., and Beese, Lorena S. Mon . "The Structure of a High Fidelity DNA Polymerase Bound to a Mismatched Nucleotide Reveals an ;Ajar; Intermediate Conformation in the Nucleotide Selection Mechanism". United States. doi:10.1074/jbc.M110.191130.
@article{osti_1021781,
title = {The Structure of a High Fidelity DNA Polymerase Bound to a Mismatched Nucleotide Reveals an ;Ajar; Intermediate Conformation in the Nucleotide Selection Mechanism},
author = {Wu, Eugene Y. and Beese, Lorena S.},
abstractNote = {To achieve accurate DNA synthesis, DNA polymerases must rapidly sample and discriminate against incorrect nucleotides. Here we report the crystal structure of a high fidelity DNA polymerase I bound to DNA primer-template caught in the act of binding a mismatched (dG:dTTP) nucleoside triphosphate. The polymerase adopts a conformation in between the previously established 'open' and 'closed' states. In this 'ajar' conformation, the template base has moved into the insertion site but misaligns an incorrect nucleotide relative to the primer terminus. The displacement of a conserved active site tyrosine in the insertion site by the template base is accommodated by a distinctive kink in the polymerase O helix, resulting in a partially open ternary complex. We suggest that the ajar conformation allows the template to probe incoming nucleotides for complementarity before closure of the enzyme around the substrate. Based on solution fluorescence, kinetics, and crystallographic analyses of wild-type and mutant polymerases reported here, we present a three-state reaction pathway in which nucleotides either pass through this intermediate conformation to the closed conformation and catalysis or are misaligned within the intermediate, leading to destabilization of the closed conformation.},
doi = {10.1074/jbc.M110.191130},
journal = {J. Biol. Chem.},
number = (22) ; 06, 2011,
volume = 286,
place = {United States},
year = {Mon Oct 10 00:00:00 EDT 2011},
month = {Mon Oct 10 00:00:00 EDT 2011}
}