Lieberman Kate R, Dahl Joseph M, Wang Hongyun
Department of Biomolecular Engineering, ‡Department of Applied Mathematics and Statistics, Baskin School of Engineering, University of California , Santa Cruz, California 95064, United States.
J Am Chem Soc. 2014 May 14;136(19):7117-31. doi: 10.1021/ja5026408. Epub 2014 May 2.
Exonucleolytic editing of incorrectly incorporated nucleotides by replicative DNA polymerases (DNAPs) plays an essential role in the fidelity of DNA replication. Editing requires that the primer strand of the DNA substrate be transferred between the DNAP polymerase and exonuclease sites, separated by a distance that is typically on the order of ~30 Å. Dynamic transitions between functional states can be quantified with single-nucleotide spatial precision and submillisecond temporal resolution from ionic current time traces recorded when individual DNAP complexes are held atop a nanoscale pore in an electric field. In this study, we have exploited this capability to determine the kinetic relationship between the translocation step and primer strand transfer between the polymerase and exonuclease sites in complexes formed between the replicative DNAP from bacteriophage Φ29 and DNA. We demonstrate that the pathway for primer strand transfer from the polymerase to exonuclease site initiates prior to the translocation step, while complexes are in the pre-translocation state. We developed a mathematical method to determine simultaneously the forward and reverse translocation rates and the rates of primer strand transfer in both directions between the polymerase and the exonuclease sites, and we have applied it to determine these rates for Φ29 DNAP complexes formed with a DNA substrate bearing a fully complementary primer-template duplex. This work provides a framework that will be extended to determine the kinetic mechanisms by which incorporation of noncomplementary nucleotides promotes primer strand transfer from the polymerase site to the exonuclease site.
复制性DNA聚合酶（DNAPs）对错误掺入的核苷酸进行核酸外切酶编辑，在DNA复制的保真度方面起着至关重要的作用。编辑要求DNA底物的引物链在DNAP聚合酶和核酸外切酶位点之间转移，这两个位点之间的距离通常在约30 Å左右。当单个DNAP复合物置于电场中的纳米级孔上方时，通过记录离子电流时间轨迹，可以以单核苷酸空间精度和亚毫秒时间分辨率对功能状态之间的动态转变进行量化。在本研究中，我们利用这一能力来确定噬菌体Φ29的复制性DNAP与DNA形成的复合物中，易位步骤与引物链在聚合酶和核酸外切酶位点之间转移的动力学关系。我们证明，引物链从聚合酶转移到核酸外切酶位点的途径在易位步骤之前就已启动，此时复合物处于易位前状态。我们开发了一种数学方法，可同时确定正向和反向易位速率以及引物链在聚合酶和核酸外切酶位点之间双向转移的速率，并将其应用于确定与携带完全互补引物 - 模板双链体的DNA底物形成的Φ29 DNAP复合物的这些速率。这项工作提供了一个框架，将进一步扩展以确定非互补核苷酸掺入促进引物链从聚合酶位点转移到核酸外切酶位点的动力学机制。
