Inactivation of the 5'-3' exonuclease of Thermus aquaticus DNA polymerase

Biochim Biophys Acta. 1995 Nov 7;1264(2):243-8. doi: 10.1016/0167-4781(95)00153-8.


The gene for Thermus aquaticus (Taq) DNA polymerase enzyme (Taq Pol I) was mutagenized and sixty-two candidate clones were screened for enzyme activity. Two of the clones expressed enzymes (*Taq-3 and *Taq-5) that showed very reduced 5'-3' exonuclease activity and normal DNA polymerase activity. These two enzymes showed heat resitance and storage stability similar to Taq Pol I and had similar effectiveness in PCR. Processivity of the polymerases was compared by measuring the extension of an end-labeled primer annealed to a single stranded DNA, as well as by a PCR method. The processivity of *Taq-3 and *Taq-5 was similar to Taq Pol I (50-80 nucleotides) and more processive than a Taq Pol I deficient in the 5'-3' exonuclease due to absence of the first 290 amino acids (Stoffel fragment). The results indicate two amino acids which are required for normal 5'-3' exonuclease activity in Taq Pol I (Arg-25 and Arg-74).

Publication types

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

MeSH terms

  • Cloning, Molecular
  • DNA Primers
  • DNA-Directed DNA Polymerase / biosynthesis
  • DNA-Directed DNA Polymerase / metabolism*
  • Enzyme Stability
  • Exodeoxyribonuclease V
  • Exodeoxyribonucleases / antagonists & inhibitors
  • Exodeoxyribonucleases / biosynthesis
  • Exodeoxyribonucleases / metabolism*
  • Genes, Bacterial
  • Mutagenesis
  • Nucleic Acid Synthesis Inhibitors
  • Recombinant Proteins / antagonists & inhibitors
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / metabolism
  • Restriction Mapping
  • Taq Polymerase
  • Thermus / enzymology*
  • Thermus / genetics


  • DNA Primers
  • Nucleic Acid Synthesis Inhibitors
  • Recombinant Proteins
  • Taq Polymerase
  • DNA-Directed DNA Polymerase
  • Exodeoxyribonucleases
  • Exodeoxyribonuclease V