Characterization of a DNA polymerase from the hyperthermophile archaea Thermococcus litoralis. Vent DNA polymerase, steady state kinetics, thermal stability, processivity, strand displacement, and exonuclease activities

J Biol Chem. 1993 Jan 25;268(3):1965-75.


We have isolated, cloned, and characterized a DNA polymerase from the hyperthermophile archaea Thermococcus litoralis, the Tli DNA polymerase (also referred to as Vent DNA polymerase). The enzyme is extremely thermostable, having a half-life of 8 h at 95 degrees C and about 2 h at 100 degrees C. Pseudo-first-order kinetics at 70 degrees C reveal an extremely low Km for a primed M13mp18 substrate (0.1 nM), coupled with a relatively high Km for dNTPs (50 microM). Accompanying extension rates are on the order of 1000 nucleotides/min. Synthesis by the polymerase is largely distributive, adding an average of 7 nucleotides/initiation event. This distributive synthesis can generate products of at least 10,000 bases. Tli DNA polymerase contains a 3'-->5' exonuclease activity that enhances the fidelity of replication by the enzyme (Mattila, P., Korpela, J., Tenkanen, T. and Pitkanen, K. (1991) Nucleic Acids Res. 19, 4967-4973). A 2-amino acid substitution within the conserved exonuclease domain abolishes both double and single strand-dependent exonuclease activity, without altering kinetic parameters for polymerization on a primed single-stranded template. Strand displacement activity by the mutated and unmutated forms increases with increasing temperature and is enhanced in the exonuclease-deficient form of the enzyme.

Publication types

  • Comparative Study

MeSH terms

  • Archaea / enzymology*
  • Base Sequence
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / isolation & purification
  • DNA-Directed DNA Polymerase / metabolism*
  • Enzyme Stability
  • Escherichia coli / genetics
  • Hot Temperature*
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism


  • Recombinant Proteins
  • Tli polymerase
  • DNA-Directed DNA Polymerase

Associated data

  • GENBANK/M74198