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 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 degrees C and it had a half-life of 2 min at 90 degrees 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 degrees C and it had a half-life of 11 min at 90 degrees C. It was usable for DNA sequencing. This is the first thermostable DNA polymerase described with the O-helix Phe-->Tyr substitution.
Blöndal
T
T
Institute of Biology, University of Iceland, Grensásvegur 12, IS-108 Reykjavik, Iceland.
Thorbjarnardóttir
S H
SH
Kieleczawa
J
J
Hjörleifsdóttir
S
S
Kristjánsson
J K
JK
Einarsson
J M
JM
Eggertsson
G
G
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology; D003001:Cloning, Molecular; D004256:DNA Polymerase I / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D020033:Protein Isoforms; D012194:RNA-Directed DNA Polymerase; D004256:DNA Polymerase I; D005092:Exonucleases