Esteban J A, Salas M, Blanco L
Centro de Biología Molecular (CSIC-UAM), Universidad Autónoma, Madrid, Spain.
J Biol Chem. 1993 Feb 5;268(4):2719-26.
Phi 29 DNA polymerase is able to catalyze two different synthetic reactions: protein-primed initiation and DNA polymerization. We have studied the fidelity of phi 29 DNA polymerase when carrying out these two reactions. Global fidelity was dissected into three steps: insertion discrimination, mismatch elongation, and proofreading. The insertion discrimination of phi 29 DNA polymerase in DNA polymerization ranged from 10(4) to 10(6). The efficiency of mismatch elongation was 10(5)-10(-6)-fold lower than that of a properly paired primer terminus. These factors indicate that DNA polymerization catalyzed by phi 29 DNA polymerase is a highly accurate process. Conversely, the insertion fidelity of protein-primed initiation was quite low, the insertion discrimination factor being about 10(2). Mismatch elongation discrimination was also rather low: mismatched terminal protein (TP).dNMP complexes were elongated from 2- to 6-fold more slowly than the correct TP.dNMP complex. Even more, the 3'-->5' exonuclease activity of phi 29 DNA polymerase was unable to act on the TP.dNMP initiation complex, precluding the possibility that a wrong dNMP covalently linked to TP could be excised and corrected. Therefore, protein-primed initiation can be predicted as a quite inaccurate reaction. The problem of maintaining the sequence at the DNA ends is discussed in the context of a recently described model for protein-primed initiation.
Phi 29 DNA聚合酶能够催化两种不同的合成反应：蛋白质引发的起始反应和DNA聚合反应。我们研究了Phi 29 DNA聚合酶在进行这两种反应时的保真度。整体保真度被细分为三个步骤：插入识别、错配延伸和校对。Phi 29 DNA聚合酶在DNA聚合反应中的插入识别范围为10^4至10^6。错配延伸的效率比正确配对的引物末端低10^5至10^-6倍。这些因素表明，Phi 29 DNA聚合酶催化的DNA聚合反应是一个高度精确的过程。相反，蛋白质引发起始反应的插入保真度相当低，插入识别因子约为10^2。错配延伸识别也相当低：错配的末端蛋白（TP）.dNMP复合物的延伸速度比正确的TP.dNMP复合物慢2至6倍。甚至，Phi 29 DNA聚合酶的3'→5'核酸外切酶活性无法作用于TP.dNMP起始复合物，排除了与TP共价连接的错误dNMP被切除和校正的可能性。因此，可以预测蛋白质引发的起始反应是一个相当不准确的反应。在最近描述的蛋白质引发起始反应模型的背景下，讨论了维持DNA末端序列的问题。
