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Biotechnology and Applied Biochemistry (2001) 34, (37–45) (Printed in Great Britain)

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Cloning, sequence analysis and functional characterization of DNA polymerase I from the thermophilic eubacterium Rhodothermus marinus

Thórarinn Blöndal*¹, Sigrídur H. Thorbjarnardóttir*, Jan Kieleczawa†, Sigrídur Hjörleifsdóttir‡², Jakob K. Kristjánsson*‡², Jón M. Einarsson§ and Gudmundur Eggertsson*³

*Institute of Biology, University of Iceland, Grensásvegur 12, IS-108 Reykjavik, Iceland, †Department of Biology, Brookhaven National Laboratory, Long Island, NY, U.S.A., ‡Department of Biotechnology, Technological Institute of Iceland, Keldnaholt, IS-110 Reykjavik, Iceland, and §GENIS, Hólmaslód 2, IS-101 Reykjavik, Iceland

Key words: dideoxynucleotide incorporation, DNA sequencing, thermostability.

Abbreviations used: ddNTP, dideoxynucleotide; ORF, open reading frame; Ni-NTA, Ni²⁺-nitrilotriacetate.

¹Present address: DeCODE Genetics Inc., Lyngháls 1, IS-110 Reykjavik, Iceland.

²Present address: Prokaria, Gylfaflöt 5, IS-112, Reykjavik, Iceland.

³To whom correspondence should be addressed (e-mail gudmegg@rhi.hi.is).

The nucleotide sequences reported here have been submitted to the GenBank^® Nucleotide Sequence Database under accession numbers AF028719 and AF028721.

A gene encoding a DNA polymerase I from the thermophilic eubacterium Rhodothermus marinus was identified. The gene was cloned, sequenced and expressed in Escherichia coli. The gene is 2772 bp long and encodes a protein of 924 amino acids with a calculated molecular mass of 104.8 kDa. Sequence analysis showed that a generally conserved Phe residue in the O-helix is substituted by a Tyr (position 756) in the R. marinus enzyme. A Tyr in this position decreases the discrimination against dideoxynucleotides which is a major advantage in DNA sequencing. The protein was purified, characterized and showed to contain specific DNA-polymerization activity of 3100 units/mg of protein, 5´ 3´ exonuclease activity and a 3´ 5´ proofreading activity. Its optimum activity was at 55 °C and it had a half-life of 2 min at 90 °C. A truncated form of the enzyme lacking the 5´ 3´ exonuclease domain was also expressed in E. coli. It had a specific DNA-polymerization activity of 5000 units/mg of protein and lacked the 5´ 3´ exonuclease activity. Its optimum activity was at 65 °C and it had a half-life of 11 min at 90 °C. It was usable for DNA sequencing. This is the first thermostable DNA polymerase described with the O-helix Phe Tyr substitution.

Received 9 April 2001/1 January 2000; accepted 27 April 2001

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