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

Abstract

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.