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

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 110 000–120 000. The three of them are devoid of exonuclease activity. Species A, B and C differ in their sensitivity towards N‐ethylmaleimide (A > B > C) and urea (A > B = C) and also in their stability at high temperature (90°C) (B > C > 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°C with enzyme A and at 63°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) · (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°C (enzyme C) and 40°C (enzyme B), while with poly(dA‐dT) they both work best at 63°C. In turn, only enzyme C is able to utilize poly(rA) · (dT)₁₀, although only with Mn²⁺ as a cofactor.