The proofreading pathway of bacteriophage T4 DNA polymerase

J Biol Chem. 1998 Sep 4;273(36):22969-76. doi: 10.1074/jbc.273.36.22969.


The base analog, 2-aminopurine (2AP), was used as a fluorescent reporter of the biochemical steps in the proofreading pathway catalyzed by bacteriophage T4 DNA polymerase. "Mutator" DNA polymerases that are defective in different steps in the exonucleolytic proofreading pathway were studied so that transient changes in fluorescence intensity could be equated with specific reaction steps. The G255S- and D131N-DNA polymerases can hydrolyze DNA, the final step in the proofreading pathway, but the mutator phenotype indicates a defect in one or more steps that prepare the primer-terminus for the cleavage reaction. The hydrolysis-defective D112A/E114A-DNA polymerase was also examined. Fluorescent enzyme-DNA complexes were preformed in the absence of Mg2+, and then rapid mixing, stopped-flow techniques were used to determine the fate of the fluorescent complexes upon the addition of Mg2+. Comparisons of fluorescence intensity changes between the wild type and mutant DNA polymerases were used to model the exonucleolytic proofreading pathway. These studies are consistent with a proofreading pathway in which the protein loop structure that contains residue Gly255 functions in strand separation and transfer of the primer strand from the polymerase active center to form a preexonuclease complex. Residue Asp131 acts at a later step in formation of the preexonuclease complex.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 2-Aminopurine
  • Bacteriophage T4 / enzymology*
  • Binding Sites
  • DNA Primers
  • DNA Replication / genetics*
  • DNA, Single-Stranded / metabolism
  • DNA-Directed DNA Polymerase / drug effects
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Exonucleases / metabolism*
  • Flow Injection Analysis
  • Fluorescent Dyes
  • Heparin / pharmacology
  • Magnesium / pharmacology
  • Models, Genetic
  • Mutation
  • Oligodeoxyribonucleotides
  • Viral Proteins / drug effects
  • Viral Proteins / genetics
  • Viral Proteins / metabolism*


  • DNA Primers
  • DNA, Single-Stranded
  • Fluorescent Dyes
  • Oligodeoxyribonucleotides
  • Viral Proteins
  • gene 43 protein, Enterobacteria phage T4
  • 2-Aminopurine
  • Heparin
  • DNA-Directed DNA Polymerase
  • Exonucleases
  • Magnesium