Cloning and Characterization of a Family B DNA Polymerase from the Hyperthermophilic Crenarchaeon Pyrobaculum islandicum

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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°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 λ DNA fragments of up to 1,500 bp.