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Applied Biochemistry and Microbiology, Vol. 41, No. 1, 2005, pp. 34–41. Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 1, 2005, pp. 40–47. Original Russian Text Copyright © 2005 by Slobodkina, Chernykh, Lopatin, Il’ina, Bannikova, Ankenbauer, El’darov, Varlamov, Bonch-Osmolovskaya.

Isolation and Characterization of Thermostable DNA Polymerase of the HyperthermophilicArchaeum Thermococcus litoralis Sh1AM
G. B. Slobodkina1, N. A. Chernykh1, S. A. Lopatin2, A. V. Il’ina2, G. E. Bannikova2, W. Ankenbauer3, M. A. El’darov2, V. P. Varlamov2, and E. A. Bonch-Osmolovskaya1
1 Institute

of Microbiology, Russian Academy of Sciences, Moscow, 117312 Russia e-mail: gslobodkina@mail.ru 2 Bioengineering Center, Russian Academy of Sciences, Moscow, 117312 Russia e-mail:varlamov@biengi.ac.ru 3 Roche Diagnostics, Mannheim, 68305 Germany e-mail: Waltraud.Ankenbauer@roche.com
Received February 20, 2004

Abstract—Overall, 30 strains of hyperthermophilic archaea, representing seven species of the genera Thermococcus, Desulfurococcus, Thermoproteus, and Acidilobus, were tested for the presence of thermostable DNA polymerases. Thermostabilities of the polymerases varieddistinctly among the strains within one species. Polymerases of five strains retained 60–100% activity upon incubation of the preparations at 95°C for 120 min. A new DNA polymerase was isolated from the strain Thermococcus litoralis Sh1AM, possessing the enzyme with the most promising properties, and characterized. Molecular weight of the enzyme is 90–100 kDa. The purified DNA polymerase preserved50% of the initial activity upon incubation at 95°C for 120 min. The polymerase isolated displayed an associated 3'-5' exonuclease activity. The error rate when extending a DNA strand was at least twofold lower compared with Taq polymerase. The main physicochemical and enzymatic properties of the new polymerase are similar to the known DNA polymerases of family B.

DNA polymerases of eubacteriaand eukaryotes are conventionally divided into the families A, B, and C, based on homology of the amino acid sequences of their catalytic subunit to Escherichia coli DNA polymerases I, II, and III, respectively. All the rest of the polymerases, including eukaryotic β-polymerase and terminal transferase, are united into family X [1]. The DNA replications of Eukarya and Bacteria are studied insufficient detail [2]; however, information on the replication system of Archaea is rather limited [3]. Virtually all the archaeal DNA polymerases known so far belong to family B [4]. Recent studies detected distinctions in the replication mechanisms of Euryarchaeota and Crenarchaeota. For example, the nucleotide sequence homologous to heterodimeric DNA polymerase of the Euryarchaeota representativePyrococcus furiosus [5] was not detected in Crenarchaeota. In addition, all the genomes of Euryarchaeota known so far encode only one DNA polymerase [6], whereas two [7, 8] and three [9] enzymes with polymerase activity were discovered in microorganisms belonging to the kingdom Crenarchaeota. Phylogenetic analysis demonstrated that crenarchaeotal polymerases formed three clusters in family B, describedas groups B1, B2, and B3 [9].

With some exceptions (Halobacterium halobium, Methanococcus vannieli, Methanococcus voltae, and Cenarchaeum symbiosum), archaeal DNA polymerases were isolated or cloned from hyperthermophilic microorganisms. Since the discovery and characterization of DNA polymerases of thermophilic microorganisms [10, 11] and their application in polymerase chain reaction [12],the thermostable polymerases became perhaps the most widespread and popular class of enzymes used in modern molecular biology, genetics, molecular diagnostics, DNA sequencing, etc. Along with the best known thermophilic DNA polymerase—Taq polymerase—archaeal DNA polymerases, which possess such advantages as increased thermostability, fidelity (due to the presence of associated 3'-5' endonuclease...