Taq酶体外合成DNA时模板错位机制的探讨

Preliminary study on the template misalignment mechanism of DNA synthesis in vitro by Taq polymerase

利用Taq DNA聚合酶体外合成DNA过程中,当反应体系中缺少与模板链互补配对的dNTP底物时,产物合成并不会在底物缺失位点处终止,聚合反应继续进行.为研究此复制缺陷现象,设计一系列模板用于DNA体外酶促合成.除了已知的碱基错配机制,笔者发现存在另一种"模板错位"机制,即模板中与底物非Watson-Crick互补配对的碱基位点首先进行收缩滑动,形成模板bulge结构后再继续进行酶促合成反应.这项研究有助于提高DNA样品合成保真度以及继续深入探索体外DNA合成的详细机制.

It is observed that the DNA polymerization in vitro catalyzed by Taq DNA polymerase does not terminate at the site when a complementary dNTP substrate is deficient, rather it keeps. To study the phenomenon of replication defect, the authors designed a series of templates for enzymatically catalyzed sy＇nthesis. In addition to the common mismatch mechanism, ＂the template misalignment mechanism＂ was proposed. It turns out that, whenever encountering at a site without a correct complementary dNTP to pair, Taq DNA polymerase at first facilitated the site mentioned above to be bulged out by the contraction and backward slippage of template, subsequently the resulting misaligned template allowed the polymerization proceeded continuously. This study is expected to have an impact on the fidelity improvement of DNA synthesis in vitro and further mechanism investigation in details.