DNA polymerization in the absence of exonucleolytic proofreading: in vivo and in vitro studies

Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2417-21. doi: 10.1073/pnas.88.6.2417.


Classical genetic selection was combined with site-directed mutagenesis to study bacteriophage T4 DNA polymerase 3'----5' exonuclease activity. A mutant DNA polymerase with very little (less than or equal to 1%) 3'----5' exonuclease activity was generated. In vivo, the 3'----5' exonuclease-deficient DNA polymerase produced the highest level of spontaneous mutation observed in T4, 500- to 1800-fold above that of wild type. The large reduction in 3'----5' exonuclease activity appears to be due to two amino acid substitutions: Glu-191 to Ala and Asp-324 to Gly. Protein sequence similarities have been observed between sequences in the Escherichia coli DNA polymerase I 3'----5' exonuclease domain and conserved sequences in eukaryotic, viral, and phage DNA polymerases. It has been proposed that the conserved sequences contain metal ion binding ligands that are required for 3'----5' exonuclease activity; however, we find that some proposed T4 DNA polymerase metal binding residues are not essential for 3'----5' exonuclease activity. Thus, our T4 DNA polymerase studies do not support the hypothesis by Bernad et al. [Bernad, A., Blanco, L., Lazaro, J.M., Martin, G. & Salas, M. (1989) Cell 59, 219-228] that many DNA polymerases, including T4 DNA polymerase, share an extensively conserved 3'----5' exonuclease motif. Therefore, extrapolation from E. coli DNA polymerase I sequence and structure to other DNA polymerases for which there is no structural information may not be valid.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • DNA Replication*
  • DNA, Viral / genetics*
  • DNA-Directed DNA Polymerase / genetics*
  • Escherichia coli / enzymology
  • Escherichia coli / genetics*
  • Exodeoxyribonuclease V
  • Exodeoxyribonucleases / genetics*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed*
  • Selection, Genetic
  • Sequence Homology, Nucleic Acid
  • T-Phages / enzymology
  • T-Phages / genetics*
  • Templates, Genetic


  • DNA, Viral
  • DNA-Directed DNA Polymerase
  • Exodeoxyribonucleases
  • Exodeoxyribonuclease V