Park Y, Choi H, Lee D S, Kim Y
School of Chemical Engineering & Technology, Yeungnam University, Kyungsan, Korea.
Mol Cells. 1997 Jun 30;7(3):419-24.
Taq DNA polymerase from Thermus aquaticus has been shown to be very useful in the polymerase chain reaction method. Taq DNA polymerase has a domain at its amino terminus (residue 1 to 291) that has a 5'-3' exonuclease activity, a 3'-5' exonuclease domain in the middle (residue 292 to 423), and a domain at its C-terminus that catalyzes polymerase reactions. Taq DNA polymerase is classified into the polI family which is represented by E. coli DNA polymerase I. The three dimensional structural alignment of 3'-5' exonuclease domains from the polI family, DNA polymerases leads us to understand why Taq DNA polymerase does not carry out proof-reading in the polymerase chain reaction. Three sequence motifs, called ExoI, II, and III must be present in order to carry out proof-reading by the 3'-5' exonuclease reaction in DNA polymerization, but Taq DNA polymerase contains none of them. The key catalytic module in the 3'-5' exonuclease is two metal ions chelated by active-site carboxylic amino acids. In order to render the 3'-5' exonuclease activity in Taq DNA polymerase, a catalytic module was constructured in the active site by protein engineering. The mutant Taq DNA polymerase shows twice as much the 3'-5' exonuclease activity as that of wild-type DNA polymerase.
已证明来自嗜热水生栖热菌的Taq DNA聚合酶在聚合酶链反应方法中非常有用。Taq DNA聚合酶在其氨基末端（第1至291位氨基酸残基）有一个结构域，具有5'-3'核酸外切酶活性，在中间（第292至423位氨基酸残基）有一个3'-5'核酸外切酶结构域，在其C末端有一个催化聚合反应的结构域。Taq DNA聚合酶被归类为以大肠杆菌DNA聚合酶I为代表的polI家族。对polI家族DNA聚合酶的3'-5'核酸外切酶结构域进行三维结构比对，有助于我们理解为什么Taq DNA聚合酶在聚合酶链反应中不进行校对。为了在DNA聚合过程中通过3'-5'核酸外切酶反应进行校对，必须存在三个序列基序，称为ExoI、II和III，但Taq DNA聚合酶中一个都没有。3'-5'核酸外切酶中的关键催化模块是由活性位点羧基氨基酸螯合的两个金属离子。为了使Taq DNA聚合酶具有3'-5'核酸外切酶活性，通过蛋白质工程在活性位点构建了一个催化模块。突变型Taq DNA聚合酶的3'-5'核酸外切酶活性是野生型DNA聚合酶的两倍。
