DNA polymerases from the extremely thermophilic bacterium Thermus thermophilus HB-8

Eur J Biochem. 1985 May 15;149(1):41-6. doi: 10.1111/j.1432-1033.1985.tb08890.x.

Abstract

Three DNA polymerase isoenzymes, which have been called A, B and C, were purified from Thermus thermophilus HB-8. These enzymes can be separated by chromatography (pH 7.5) on phosphocellulose and DNA-agarose. Their relative molecular masses, as determined by glycerol gradient centrifugation, fall in the range of 110000-120000. The three of them are devoid of exonuclease activity. Species A, B and C differ in their sensitivity towards N-ethylmaleimide (A greater than B greater than C) and urea (A greater than B = C) and also in their stability at high temperature (90 degrees C) (B greater than C greater than A). In addition, these enzymes can be distinguished utilizing various templates under different conditions. Thus, with activated DNA and Mg2+ as a cofactor, the highest incorporation is obtained at 50 degrees C with enzyme A and at 63 degrees C with enzymes B and C. If Mg2+ is replaced by Mn2+, the optimal temperatures remain unchanged, but enzyme A is stimulated twofold, while the activities of enzymes B and C decrease to one-half. On the other hand, with either poly(dA) X (dT)10 or poly(dA-dT) and Mg2+, enzyme A is inactive and enzyme C is severalfold more active than enzyme B. With the former synthetic template, optimal temperatures are 50 degrees C (enzyme C) and 40 degrees C (enzyme B), while with poly(dA-dT) they both work best at 63 degrees C. In turn, only enzyme C is able to utilize poly(rA) X (dT)10, although only with Mn2+ as a cofactor.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Centrifugation, Density Gradient
  • Chemical Phenomena
  • Chemistry
  • Chromatography, Affinity
  • DNA-Directed DNA Polymerase / classification
  • DNA-Directed DNA Polymerase / isolation & purification*
  • Ethylmaleimide / pharmacology
  • Hot Temperature
  • Molecular Weight
  • Substrate Specificity
  • Thermus / enzymology*
  • Urea / pharmacology

Substances

  • Urea
  • DNA-Directed DNA Polymerase
  • Ethylmaleimide