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

A gene encoding a DNA polymerase I from the thermophilic eubacterium Rhodot hermus 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 analys is showed that a generally conserved Phe residue in the O-helix is substitu ted by a Tyr (position 756) in the R. marinus enzyme. A Tyr in this positio n decreases the discrimination against dideoxynucleoticles which is a major advantage in DNA sequencing. The protein was purified, characterized and s howed to contain specific DNA-polymerization activity of 3 100 units/mg of protein, 5 ' --> 3 ' exonuclease activity and a 3 ' --> 5 ' proofreading ac tivity. Its optimum activity was at 55 degreesC and it had a half-life of 2 min at 90 degreesC. A truncated form of the enzyme lacking the 5 ' --> 3 ' exonuclease domain was also expressed in E. coli. It had a specific DNA-po lymerization activity of 5000 units/mg of protein and lacked the 5 ' --> 3 ' exonuclease activity. Its optimum activity was at 65 degreesC and it had a half-life of 11 min at 90 degreesC. It was usable for DNA sequencing. Thi s is the first thermostable DNA polymerase described with the O-helix Phe - -> Tyr substitution.