Thermostable DNA polymerase from the archaebacterium Sulfolobus acidocaldarius. Purification, characterization and immunological properties.
We have purified to near homogeneity a DNA polymerase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme revealed a polypeptide of 100 kDa. On the basis of a Stokes radius of 4.2 nm and a sedimentation coefficient of 6 S, the purified enzyme has an estimated molecular mass of 109 kDa. These results are consistent with the enzyme being a monomer of 100 kDa. In addition a polyclonal antiserum, obtained by injection of the electroeluted 100-kDa polypeptide into a rabbit, specifically neutralized the DNA-polymerase activity. The enzyme is sensitive to both N-ethylmaleimide and 2',3'-dideoxyribosylthymine triphosphate and resistant to aphidicolin. The purified DNA polymerase has neither exonuclease nor primase activities. In our in vitro conditions, the enzyme is thermostable up to 80 degrees C and is active between 55 degrees C and 85 degrees C in the presence of activated calf-thymus DNA.
Elie
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C
Groupe de Biologie et Génétique Moléculaires, Centre National de la Recherche Scientifique, Villejuif, France.
De Recondo
A M
AM
Forterre
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P
D001105:Archaea / Q000201:enzymology*; D001419:Bacteria / Q000201:enzymology*; D002499:Centrifugation, Density Gradient / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D006358:Hot Temperature; D007202:Indicators and Reagents; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D013698:Templates, Genetic
D007202:Indicators and Reagents; D046911:Macromolecular Substances; D004259:DNA-Directed DNA Polymerase