Bukhrashvili I S, Chinchaladze D Z, Lavrik O I, Levina A S, Nevinsky G A, Prangishvili D A
Institute of Molecular Biology and Biological Physics, Georgian SSR Academy of Sciences, Tbilisi, U.S.S.R.
Biochim Biophys Acta. 1989 Jun 1;1008(1):102-7. doi: 10.1016/0167-4781(89)90175-9.
Optimal conditions for polymerization reaction catalyzed on poly(dA) and poly(dT) templates by DNA polymerases from thermoacidophilic archaebacteria--DNA polymerase A from Sulfolobus acidocaldarius and DNA polymerase B from Thermoplasma acidophilum--have been established. Values of Km and Vmax (60 degrees C) for a set of primers d(pA)n and d(pT)n have been estimated. Minimal primers for both enzymes are dNMP. Lengthening of primers by each mononucleotide increases their affinity about 2.16-fold. Linear dependence of log Km and of log vmax on the number of mononucleotide links in primers (n) has breaking point at n = 10. The value of Vmax is about 20% of that for decanucleotide. The affinity of the primer d(pA)9p(rib*) with a deoxyribosylurea residue at the 3'-end does not differ essentially from that of d(pA)9. Substitution of the 3'-terminal nucleotide of a complementary primer for a noncomplementary nucleotide, e.g., substitution of 3'-terminal A for C in d(pA)10 in the reaction catalyzed on poly(dT), decreases the affinity of a primer by one order of magnitude.
已确定嗜热嗜酸古细菌的DNA聚合酶（来自嗜酸硫化叶菌的DNA聚合酶A和嗜热嗜酸栖热菌的DNA聚合酶B）在聚（dA）和聚（dT）模板上催化聚合反应的最佳条件。已估算出一组引物d（pA）n和d（pT）n的Km和Vmax（60℃）值。两种酶的最小引物都是dNMP。每个单核苷酸使引物延长会使其亲和力增加约2.16倍。引物中log Km和log vmax对单核苷酸连接数（n）的线性依赖性在n = 10处有断点。Vmax值约为十聚体的20%。3'-末端带有脱氧核糖基脲残基的引物d（pA）9p（rib*）的亲和力与d（pA）9基本无差异。在聚（dT）上催化的反应中，将互补引物的3'-末端核苷酸替换为非互补核苷酸，例如在d（pA）10中将3'-末端的A替换为C，会使引物的亲和力降低一个数量级。
