Mismatch-induced conformational distortions in polymerase β support an induced-fit mechanism for fidelity

Karunesh Arora, William A. Beard, Samuel H. Wilson, Tamar Schlick

Research output: Contribution to journalArticle

Abstract

Molecular dynamics simulations of DNA polymerase (pol) β complexed with different incorrect incoming nucleotides (G·G, G·T, and T·T template base-incoming nucleotide combinations) at the template-primer terminus are analyzed to delineate structure-function relationships for aberrant base pairs in a polymerase active site. Comparisons, made to pol β structure and motions in the presence of a correct base pair, are designed to gain atomically detailed insights into the process of nucleotide selection and discrimination. In the presence of an incorrect incoming nucleotide, α-helix N of the thumb subdomain believed to be required for pol β' s catalytic cycling moves toward the open conformation rather than the closed conformation as observed for the correct base pair (G·C) before the chemical reaction. Correspondingly, active-site residues in the microenvironment of the incoming base are in intermediate conformations for non-Watson-Crick pairs. The incorrect incoming nucleotide and the corresponding template residue assume distorted conformations and do not form Watson-Crick bonds. Furthermore, the coordination number and the arrangement of ligands observed around the catalytic and nucleotide binding magnesium ions are mismatch specific. Significantly, the crucial nucleotidyl transferase reaction distance (Pα-O3′) for the mismatches between the incoming nucleotide and the primer terminus is not ideally compatible with the chemical reaction of primer extension that follows these conformational changes. Moreover, the extent of active-site distortion can be related to experimentally determined rates of nucleotide misincorporation and to the overall energy barrier associated with polymerase activity. Together, our studies provide structure-function insights into the DNA polymerase-induced constraints (i.e., α-helix N conformation, DNA base pair bonding, conformation of protein residues in the vicinity of dNTP, and magnesium ions coordination) during nucleotide discrimination and pol β-nucleotide interactions specific to each mispair and how they may regulate fidelity. They also lend further support to our recent hypothesis that additional conformational energy barriers are involved following nucleotide binding but prior to the chemical reaction.

Original languageEnglish (US)
Pages (from-to)13328-13341
Number of pages14
JournalBiochemistry
Volume44
Issue number40
DOIs
StatePublished - Oct 11 2005

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Nucleotides
Conformations
Base Pairing
Chemical reactions
Catalytic Domain
Energy barriers
DNA-Directed DNA Polymerase
Magnesium
Ions
Nucleic Acid Conformation
Protein Conformation
Thumb
Molecular Dynamics Simulation
Transferases
Molecular dynamics
Ligands
DNA
Computer simulation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mismatch-induced conformational distortions in polymerase β support an induced-fit mechanism for fidelity. / Arora, Karunesh; Beard, William A.; Wilson, Samuel H.; Schlick, Tamar.

In: Biochemistry, Vol. 44, No. 40, 11.10.2005, p. 13328-13341.

Research output: Contribution to journalArticle

Arora, Karunesh ; Beard, William A. ; Wilson, Samuel H. ; Schlick, Tamar. / Mismatch-induced conformational distortions in polymerase β support an induced-fit mechanism for fidelity. In: Biochemistry. 2005 ; Vol. 44, No. 40. pp. 13328-13341.
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abstract = "Molecular dynamics simulations of DNA polymerase (pol) β complexed with different incorrect incoming nucleotides (G·G, G·T, and T·T template base-incoming nucleotide combinations) at the template-primer terminus are analyzed to delineate structure-function relationships for aberrant base pairs in a polymerase active site. Comparisons, made to pol β structure and motions in the presence of a correct base pair, are designed to gain atomically detailed insights into the process of nucleotide selection and discrimination. In the presence of an incorrect incoming nucleotide, α-helix N of the thumb subdomain believed to be required for pol β' s catalytic cycling moves toward the open conformation rather than the closed conformation as observed for the correct base pair (G·C) before the chemical reaction. Correspondingly, active-site residues in the microenvironment of the incoming base are in intermediate conformations for non-Watson-Crick pairs. The incorrect incoming nucleotide and the corresponding template residue assume distorted conformations and do not form Watson-Crick bonds. Furthermore, the coordination number and the arrangement of ligands observed around the catalytic and nucleotide binding magnesium ions are mismatch specific. Significantly, the crucial nucleotidyl transferase reaction distance (Pα-O3′) for the mismatches between the incoming nucleotide and the primer terminus is not ideally compatible with the chemical reaction of primer extension that follows these conformational changes. Moreover, the extent of active-site distortion can be related to experimentally determined rates of nucleotide misincorporation and to the overall energy barrier associated with polymerase activity. Together, our studies provide structure-function insights into the DNA polymerase-induced constraints (i.e., α-helix N conformation, DNA base pair bonding, conformation of protein residues in the vicinity of dNTP, and magnesium ions coordination) during nucleotide discrimination and pol β-nucleotide interactions specific to each mispair and how they may regulate fidelity. They also lend further support to our recent hypothesis that additional conformational energy barriers are involved following nucleotide binding but prior to the chemical reaction.",
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