Construction and characterization of a bacteriophage T4 DNA polymerase deficient in 3'-->5' exonuclease activity

Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2579-83. doi: 10.1073/pnas.90.7.2579.


Bacteriophage T4 DNA polymerase has a proofreading 3'-->5' exonuclease that plays an important role in maintaining the accuracy of DNA replication. We have constructed a T4 DNA polymerase deficient in this exonuclease by converting Asp-219 to Ala. The exonuclease activity of the mutant T4 DNA polymerase has been reduced by a factor of at least 10(7), but it retains a polymerase activity whose kinetic parameters, kcat, Kd DNA, and Kd dATP, are very close to those of the wild-type enzyme. Bacteriophage T4 with the mutant polymerase gene has a markedly increased mutation frequency. Asp-219 in T4 DNA polymerase is within a sequence similar to those surrounding Asp residues previously shown to be essential for the exonuclease activities of the Klenow fragment of Escherichia coli DNA polymerase I (Asp-424), bacteriophage phi 29 DNA polymerase (Asp-66), and Saccharomyces cerevisiae DNA polymerase delta (Asp-405). Thus, these studies support the proposal that there are similar sequences in the active sites for the proofreading exonucleases of these and related DNA polymerases.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Bacteriophage T4 / enzymology*
  • Bacteriophage T4 / genetics
  • Base Sequence
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Deoxyadenine Nucleotides / metabolism
  • Escherichia coli / genetics
  • Exodeoxyribonuclease V
  • Exodeoxyribonucleases / genetics
  • Exodeoxyribonucleases / metabolism*
  • Genes, Viral
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Oligodeoxyribonucleotides
  • Promoter Regions, Genetic
  • Protein Binding
  • Restriction Mapping


  • Deoxyadenine Nucleotides
  • Oligodeoxyribonucleotides
  • DNA-Directed DNA Polymerase
  • Exodeoxyribonucleases
  • Exodeoxyribonuclease V
  • 2'-deoxyadenosine triphosphate