Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion

Nat Commun. 2013;4:2131. doi: 10.1038/ncomms3131.

Abstract

The fidelity of DNA polymerases depends on conformational changes that promote the rejection of incorrect nucleotides before phosphoryl transfer. Here, we combine single-molecule FRET with the use of DNA polymerase I and various fidelity mutants to highlight mechanisms by which active-site side chains influence the conformational transitions and free-energy landscape that underlie fidelity decisions in DNA synthesis. Ternary complexes of high fidelity derivatives with complementary dNTPs adopt mainly a fully closed conformation, whereas a conformation with a FRET value between those of open and closed is sparsely populated. This intermediate-FRET state, which we attribute to a partially closed conformation, is also predominant in ternary complexes with incorrect nucleotides and, strikingly, in most ternary complexes of low-fidelity derivatives for both correct and incorrect nucleotides. The mutator phenotype of the low-fidelity derivatives correlates well with reduced affinity for complementary dNTPs and highlights the partially closed conformation as a primary checkpoint for nucleotide selection.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Catalytic Domain
  • DNA Polymerase I / chemistry*
  • DNA Polymerase I / genetics
  • DNA Polymerase I / metabolism
  • DNA Replication
  • Escherichia coli / chemistry*
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Fluorescence Resonance Energy Transfer
  • Geobacillus stearothermophilus / chemistry
  • Kinetics
  • Models, Molecular*
  • Mutagenesis, Insertional
  • Nucleotides / chemistry*
  • Nucleotides / genetics
  • Nucleotides / metabolism
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Substrate Specificity

Substances

  • Bacterial Proteins
  • Nucleotides
  • DNA Polymerase I