Purification and characterization of a DNA polymerase from the archaebacterium Thermoplasma acidophilum

Abstract

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°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°C but the exonuclease activity is active in a broader range of lower temperatures and is more thermostable than the DNA polymerase activity. Copyright © 1990, Wiley Blackwell. All rights reserved