Cloning and characterization of a family B DNA polymerase from the hyperthermophilic crenarchaeon Pyrobaculum islandicum

J Bacteriol. 2000 Feb;182(3):655-63. doi: 10.1128/JB.182.3.655-663.2000.

Abstract

In order to extend the limited knowledge about crenarchaeal DNA polymerases, we cloned a gene encoding a family B DNA polymerase from the hyperthermophilic crenarchaeon Pyrobaculum islandicum. The enzyme shared highest sequence identities with a group of phylogenetically related DNA polymerases, designated B3 DNA polymerases, from members of the kingdom Crenarchaeota, Pyrodictium occultum and Aeropyrum pernix, and several members of the kingdom Euryarchaeota. Six highly conserved regions as well as a DNA-binding motif, indicative of family B DNA polymerases, were identified within the sequence. Furthermore, three highly conserved 3'-5' exonuclease motifs were also found. The gene was expressed in Escherichia coli, and the DNA polymerase was purified to homogeneity by heat treatment and affinity chromatography. Activity staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an active polypeptide of approximately 90 kDa. For the recombinant DNA polymerase from P. islandicum, activated calf thymus DNA was used as a substrate rather than primed single-stranded DNA. The enzyme was strongly inhibited by monovalent cations and N-ethylmaleimide; it is moderately sensitive to aphidicolin and dideoxyribonucleoside triphosphates. The half-life of the enzyme at 100 and 90 degrees C was 35 min and >5 h, respectively. Interestingly, the pH of the assay buffer had a significant influence on the 3'-5' exonuclease activity of the recombinant enzyme. Under suitable assay conditions for PCR, the enzyme was able to amplify lambda DNA fragments of up to 1,500 bp.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular
  • DNA-Directed DNA Polymerase / biosynthesis*
  • DNA-Directed DNA Polymerase / genetics
  • Gene Expression Regulation, Archaeal
  • Gene Expression Regulation, Enzymologic
  • Molecular Sequence Data
  • Phylogeny
  • Polymerase Chain Reaction
  • Sequence Alignment
  • Thermoproteaceae / enzymology*
  • Thermoproteaceae / genetics

Substances

  • DNA-Directed DNA Polymerase