Purification and characterization of a DNA polymerase from the archaebacterium Thermoplasma acidophilum.
A thermophilic DNA polymerase has been purified to near homogeneity from the archaebacterium Thermoplasma acidophilum. Analysis of the purified enzyme by sodium dodecyl sulfate gel electrophoresis revealed a single polypeptide of 88 kDa which co-sediments with the DNA polymerase activity on sucrose gradients. Combination of sedimentation and gel filtration analyses indicates that this DNA polymerase is an 88-kDa monomeric enzyme in its native form. The DNA polymerase is resistant to aphidicolin, slightly sensitive to 2',3'-dideoxyribosylthymine triphosphate and inhibited by N-ethylmaleimide when preincubation with this reagent is performed at 65 degrees C. We find that a 3'----5' exonuclease activity is associated with the purified DNA polymerase; the two activities of the enzyme are optimal at 65 degrees C but the exonuclease activity is active in a broader range of lower temperatures and is more thermostable than the DNA polymerase activity.
Hamal
A
A
Institut de Génétique et Microbiologie, Université Paris-Sud, Centre d'Orsay, France.
Forterre
P
P
Elie
C
C
D001105:Archaea / Q000201:enzymology*; D001419:Bacteria / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification* / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D013698:Templates, Genetic
D046911:Macromolecular Substances; D004259:DNA-Directed DNA Polymerase