Lokhova I A, Nevinskiĭ G A, Bulychev N A, Gorn V V, Levina A S, Rudenko N K, Kavsan V M, Lavrik O I
Mol Biol (Mosk). 1990 Mar-Apr;24(2):396-407.
Optimal conditions for the reaction of polymerization catalyzed by RNA-dependent DNA-polymerase from AMV on poly(A)- and poly(dA)-templates with d(pT)n-primers were established. Optimal concentrations of the components and pH of the reaction mixtures were found out to differ significantly. dTTP was shown to be both a nucleotide substrate and a minimal primer of the polymerization. The Km values for d(pT)2-primer (Km = 0.11 mM and 0.54 for poly(A) and poly(dA)-templates, respectively) and longer oligothymidylates were estimated. The lengthening of d(pT)n (n = 2-10) by one mononucleotide unit led to a 3-fold and 2-fold decrease of Km value for poly(A) and poly(dA), respectively. Further lengthening of the primer (n = 10-25) did not affect Km for the primers. The maximal rates of polymerization did not depend on primer length. The activation reaction (Ea = 12 kcal/mol) of polymerization on poly(A) was considerably lower than that on poly(dA) (Ea = 50 kcal/mol). In both cases a highly processive polymerization was observed. It was suggested that the synthesis had been more effective on poly(A)-template due to a more effective formation of the complex enzyme primer template.
确定了禽成髓细胞瘤病毒（AMV）的RNA依赖性DNA聚合酶在以聚（A）和聚（dA）为模板、d（pT）n为引物时催化聚合反应的最佳条件。发现反应混合物各组分的最佳浓度和pH值存在显著差异。结果表明，dTTP既是聚合反应的核苷酸底物，也是最小引物。估算了d（pT）2引物（聚（A）和聚（dA）模板的Km值分别为0.11 mM和0.54）以及更长的寡聚胸苷酸的Km值。d（pT）n（n = 2 - 10）延长一个单核苷酸单位会导致聚（A）和聚（dA）的Km值分别降低3倍和2倍。引物进一步延长（n = 10 - 25）对引物的Km值没有影响。聚合反应的最大速率不取决于引物长度。聚（A）上聚合反应的活化反应（Ea = 12千卡/摩尔）明显低于聚（dA）上的活化反应（Ea = 50千卡/摩尔）。在两种情况下均观察到高度持续的聚合反应。有人认为，由于酶 - 引物 - 模板复合物形成更有效，聚（A）模板上的合成更有效。
