Journal of Bacteriology, February 2000, p. 655-663, Vol. 182, No. 3
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
andDepartment of Technical Microbiology, Technical University Hamburg-Harburg, Denickestrasse 15, D-21071 Hamburg, Germany
Received 28 June 1999/Accepted 27 October 1999
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
DNA fragments of
up to 1,500 bp.
Dedicated to Gerhard Gottschalk on his 65th birthday.
Present address: Aventis Research & Technologies GmbH & Co. KG,
Department for Operative Research, Catalysis, Industriepark Höchst, D-65926 Frankfurt/Main, Germany.
This article has been cited by other articles:
| Appl. Environ. Microbiol. | Infect. Immun. | Eukaryot. Cell |
|---|---|---|
| Mol. Cell. Biol. | J. Virol. | Microbiol. Mol. Biol. Rev. |
| ALL ASM JOURNALS |