Department of Biomolecular Engineering, University of California, Santa Cruz, Baskin School of Engineering, 1156 High Street, MS: SOE2, Santa Cruz, California 95064, USA.
J Am Chem Soc. 2013 Jun 19;135(24):9149-55. doi: 10.1021/ja403640b. Epub 2013 Jun 7.
Complexes formed between phi29 DNA polymerase (DNAP) and DNA fluctuate discretely between the pre-translocation and post-translocation states on the millisecond time scale. The translocation fluctuations can be observed in ionic current traces when individual complexes are captured atop the α-hemolysin nanopore in an electric field. The presence of complementary 2'-deoxynucleoside triphosphate (dNTP) shifts the equilibrium across the translocation step toward the post-translocation state. Here we have determined quantitatively the kinetic relationship between the phi29 DNAP translocation step and dNTP binding. We demonstrate that dNTP binds to phi29 DNAP-DNA complexes only after the transition from the pre-translocation state to the post-translocation state; dNTP binding rectifies the translocation but it does not directly drive the translocation. Based on the measured time traces of current amplitude, we developed a method for determining the forward and reverse translocation rates and the dNTP association and dissociation rates, individually at each dNTP concentration and each voltage. The translocation rates, and their response to force, match those determined for phi29 DNAP-DNA binary complexes and are unaffected by dNTP. The dNTP association and dissociation rates do not vary as a function of voltage, indicating that force does not distort the polymerase active site and that dNTP binding does not directly involve a displacement in the translocation direction. This combined experimental and theoretical approach and the results obtained provide a framework for separately evaluating the effects of biological variables on the translocation transitions and their effects on dNTP binding.
phi29 DNA 聚合酶（DNAP）与 DNA 形成的复合物在毫秒时间尺度上在预迁移和后迁移状态之间离散波动。当单个复合物在电场中被捕获在α-溶血素纳米孔顶部时，可以在离子电流迹线中观察到迁移波动。互补的 2'-脱氧核苷三磷酸（dNTP）的存在将平衡转移到后迁移状态。在这里，我们定量确定了 phi29 DNAP 迁移步骤与 dNTP 结合之间的动力学关系。我们证明 dNTP 仅在从预迁移状态过渡到后迁移状态后才与 phi29 DNAP-DNA 复合物结合；dNTP 结合纠正了迁移，但它不会直接驱动迁移。基于电流幅度的测量时间轨迹，我们开发了一种方法，用于单独确定每个 dNTP 浓度和每个电压下的正向和反向迁移率以及 dNTP 结合和解离率。迁移率及其对力的响应与 phi29 DNAP-DNA 二元复合物的迁移率相匹配，并且不受 dNTP 的影响。dNTP 结合和解离率不随电压变化，表明力不会扭曲聚合酶活性位点，并且 dNTP 结合不会直接涉及迁移方向的位移。这种组合的实验和理论方法以及获得的结果为分别评估生物变量对迁移转变及其对 dNTP 结合的影响提供了一个框架。
