Mutational and pH studies of the 3' --> 5' exonuclease activity of bacteriophage T4 DNA polymerase

J Biol Chem. 1999 Aug 27;274(35):25151-8. doi: 10.1074/jbc.274.35.25151.


The 3' --> 5' exonuclease activity of proofreading DNA polymerases requires two divalent metal ions, metal ions A and B. Mutational studies of the 3' --> 5' exonuclease active center of the bacteriophage T4 DNA polymerase indicate that residue Asp-324, which binds metal ion A, is the single most important residue for the hydrolysis reaction. In the absence of a nonenzymatic source of hydroxide ions, an alanine substitution for residue Asp-324 reduced exonuclease activity 10-100-fold more than alanine substitutions for the other metal-binding residues, Asp-112 and Asp-219. Thus, exonuclease activity is reduced 10(5)-fold for the D324A-DNA polymerase compared with the wild-type enzyme, while decreases of 10(3)- to 10(4)-fold are detected for the D219A- and D112A/E114A-DNA polymerases, respectively. Our results are consistent with the proposal that a water molecule, coordinated by metal ion A, forms a metal-hydroxide ion that is oriented to attack the phosphodiester bond at the site of cleavage. Residues Glu-114 and Lys-299 may assist the reaction by lowering the pK(a) of the metal ion-A coordinated water molecule, whereas residue Tyr-320 may help to reorient the DNA from the binding conformation to the catalytically active conformation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • DNA Polymerase I / chemistry
  • DNA-Directed DNA Polymerase*
  • Escherichia coli / enzymology
  • Exonucleases / metabolism*
  • Hydrogen-Ion Concentration
  • Metals / metabolism
  • Models, Molecular
  • Mutation
  • Nucleic Acid Conformation
  • Poly T / metabolism
  • Protein Binding
  • Protein Conformation
  • RNA, Catalytic / chemistry
  • Viral Proteins / chemistry
  • Viral Proteins / genetics*
  • Water / chemistry


  • Metals
  • RNA, Catalytic
  • Viral Proteins
  • gene 43 protein, Enterobacteria phage T4
  • Water
  • Poly T
  • DNA Polymerase I
  • DNA-Directed DNA Polymerase
  • Exonucleases