Role of a GAG Hinge in the Nucleotide-induced Conformational Change Governing Nucleotide Specificity by T7 DNA Polymerase*

A nucleotide-induced change in DNA polymerase structure governs the kinetics of polymerization by high fidelity DNA polymerases. Mutation of a GAG hinge (G542A/G544A) in T7 DNA polymerase resulted in a 1000-fold slower rate of conformational change, which then limited the rate of correct nucleotide incorporation. Rates of misincorporation were comparable to that seen for wild-type enzyme so that the net effect of the mutation was a large decrease in fidelity. We demonstrate that a presumably modest change from glycine to alanine 20 Å from the active site can severely restrict the flexibility of the enzyme structure needed to recognize and incorporate correct substrates with high specificity. These results emphasize the importance of the substrate-induced conformational change in governing nucleotide selectivity by accelerating the incorporation of correct base pairs but not mismatches.

• Publication year

  2010

• Venue

  Journal of Biological Chemistry

• Publication date

  2010-10-26

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1074/jbc.M110.156737PMID 20978284PMCID PMC3020739
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• Nucleotide-dependent conformational change governs specificity and analog discrimination by HIV reverse transcriptase

  2010cited by this paper
• Site-specific labeling of T7 DNA polymerase with a conformationally sensitive fluorophore and its use in detecting single-nucleotide polymorphisms.

  2009cited by this paper
• Theoretical studies of short polyproline systems: recalibration of a molecular ruler.

  2009cited by this paper
• Global kinetic explorer: a new computer program for dynamic simulation and fitting of kinetic data.

  2009cited by this paper
• FitSpace explorer: an algorithm to evaluate multidimensional parameter space in fitting kinetic data.

  2009cited by this paper
• Role of Induced Fit in Enzyme Specificity: A Molecular Forward/Reverse Switch*

  2008cited by this paper
• A unified kinetic mechanism applicable to multiple DNA polymerases.

  2007cited by this paper
• A novel mechanism of selectivity against AZT by the human mitochondrial DNA polymerase

  2007cited by this paper
• A new paradigm for DNA polymerase specificity.

  2006cited by this paper
• Incorporation and Replication of 8-Oxo-deoxyguanosine by the Human Mitochondrial DNA Polymerase*

  2006cited by this paper
• Motions of the fingers subdomain of klentaq1 are fast and not rate limiting: implications for the molecular basis of fidelity in DNA polymerases.

  2005cited by this paper
• A lysine residue in the fingers subdomain of T7 DNA polymerase modulates the miscoding potential of 8-oxo-7,8-dihydroguanosine.

  2005cited by this paper
• Use of 2-aminopurine fluorescence to examine conformational changes during nucleotide incorporation by DNA polymerase I (Klenow fragment).

  2003cited by this paper
• A reexamination of the nucleotide incorporation fidelity of DNA polymerases.

  2002cited by this paper
• Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 Å resolution

  1998cited by this paper
• Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation

  1998cited by this paper
• Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase.

  1996cited by this paper
• An induced-fit kinetic mechanism for DNA replication fidelity: direct measurement by single-turnover kinetics.

  1991cited by this paper
• Ribozyme-catalyzed and nonenzymatic reactions of phosphate diesters: rate effects upon substitution of sulfur for a nonbridging phosphoryl oxygen atom.

  1991cited by this paper
• Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant.

  1991cited by this paper
• The role of induced fit and conformational changes of enzymes in specificity and catalysis

  1988cited by this paper
• Kinetic mechanism of DNA polymerase I (Klenow).

  1987cited by this paper
• Rate-limiting steps in the DNA polymerase I reaction pathway.

  1985cited by this paper
• Analysis of numerical methods for computer simulation of kinetic processes: development of KINSIM--a flexible, portable system.

  1983cited by this paper
• Intermediate states of subfragment 1 and actosubfragment 1 ATPase: reevaluation of the mechanism.

  1978cited by this paper
• Enzyme structure and mechanism

  1977cited by this paper
• Bioinformatics Original Paper Copasi—a Complex Pathway Simulator

  year unknowncited by this paper

• A Change in the Rate-Determining Step of Polymerization by the K289M DNA Polymerase β Cancer-Associated Variant.

  2017cites this paper
• Rapid Time Scale Analysis of T4 DNA Ligase-DNA Binding.

  2017cites this paper
• Y-family polymerase conformation is a major determinant of fidelity and translesion specificity.

  2013cites this paper
• NTP-mediated nucleotide excision activity of hepatitis C virus RNA-dependent RNA polymerase

  2013cites this paper
• Distal structural elements coordinate a conserved base flipping network.

  2013cites this paper
• [The fidelity mechanism of DNA synthesis].

  2012cites this paper
• Assembly, Purification, and Pre-steady-state Kinetic Analysis of Active RNA-dependent RNA Polymerase Elongation Complex*

  2012cites this paper
• The fidelity mechanism of DNA synthesis: The fidelity mechanism of DNA synthesis

  2012cites this paper
• Clustering of Alpers disease mutations and catalytic defects in biochemical variants reveal new features of molecular mechanism of the human mitochondrial replicase , Pol c

  2011cites this paper
• Clustering of Alpers disease mutations and catalytic defects in biochemical variants reveal new features of molecular mechanism of the human mitochondrial replicase, Pol γ

  2011cites this paper
• Fine Tuning of a Biological Machine: DnaK Gains Improved Chaperone Activity by Altered Allosteric Communication and Substrate Binding

  2011cites this paper
• Clustering of Alpers disease mutations and catalytic defects in biochemical variants reveal new features of molecular mechanism of hte human mitochondrial replicase , Pol gamma Euro

  year unknowncites this paper