DNA polymerases from the extremely thermophilic bacterium Thermus thermophilus HB-8.
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.
D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D004259:DNA-Directed DNA Polymerase / Q000145:classification / Q000302:isolation & purification*; D005033:Ethylmaleimide / Q000494:pharmacology; D006358:Hot Temperature; D008970:Molecular Weight; D013379:Substrate Specificity; D013824:Thermus / Q000201:enzymology*; D014508:Urea / Q000494:pharmacology
D014508:Urea; D004259:DNA-Directed DNA Polymerase; D005033:Ethylmaleimide