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Journal of Bacteriology, October 1999, p. 5984-5992, Vol. 181, No. 19
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Two Family B DNA Polymerases from Aeropyrum pernix, an Aerobic Hyperthermophilic Crenarchaeote

Isaac K. O. Cann,1 Sonoko Ishino,1 Norimichi Nomura,2 Yoshihiko Sako,2 and Yoshizumi Ishino1,*

Department of Molecular Biology, Biomolecular Engineering Research Institute, Suita, Osaka 565-0874,1 and Laboratory of Marine Microbiology, Division of Applied Bioscience, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502,2 Japan

Received 16 April 1999/Accepted 29 July 1999

DNA polymerase activities in fractionated cell extract of Aeropyrum pernix, a hyperthermophilic crenarchaeote, were investigated. Aphidicolin-sensitive (fraction I) and aphidicolin-resistant (fraction II) activities were detected. The activity in fraction I was more heat stable than that in fraction II. Two different genes (polA and polB) encoding family B DNA polymerases were cloned from the organism by PCR using degenerated primers based on the two conserved motifs (motif A and B). The deduced amino acid sequences from their entire coding regions contained all of the motifs identified in family B DNA polymerases for 3'right-arrow5' exonuclease and polymerase activities. The product of polA gene (Pol I) was aphidicolin resistant and heat stable up to 80°C. In contrast, the product of polB gene (Pol II) was aphidicolin sensitive and stable at 95°C. These properties of Pol I and Pol II are similar to those of fractions II and I, respectively, and moreover, those of Pol I and Pol II of Pyrodictium occultum. The deduced amino acid sequence of A. pernix Pol I exhibited the highest identities to archaeal family B DNA polymerase homologs found only in the crenarchaeotes (group I), while Pol II exhibited identities to homologs found in both euryarchaeotes and crenarchaeotes (group II). These results provide further evidence that the subdomain Crenarchaeota has two family B DNA polymerases. Furthermore, at least two DNA polymerases work in the crenarchaeal cells, as found in euryarchaeotes, which contain one family B DNA polymerase and one heterodimeric DNA polymerase of a novel family.

* Corresponding author. Mailing address: Department of Molecular Biology, Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan. Phone: 81-6-872-8208. Fax: 81-6-872-8219. E-mail: ishino{at}beri.co.jp.

Journal of Bacteriology, October 1999, p. 5984-5992, Vol. 181, No. 19
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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