Kinetic mechanism of translocation and dNTP binding in individual DNA polymerase complexes.
Kinetic mechanism of translocation and dNTP binding in individual DNA polymerase complexes.
复制标题
DOI：
10.1021/ja403640b
复制
发表时间：
2013-06-19
影响因子：
15
通讯作者：
Wang, Hongyun
中科院分区：
文献类型：
--
作者：
Lieberman, Kate R.;Dahl, Joseph M.;Mai, Ai H.;Cox, Ashley;Akeson, Mark;Wang, Hongyun
Phi29DNA聚合酶(DNAP)与DNA形成的复合体在毫秒尺度上以离散的方式在移位前和移位后的状态之间波动。当单个络合物在电场中捕获到α-溶血素纳米孔的顶部时，可以在离子电流轨迹中观察到移位波动。互补的2‘-脱氧核苷三磷酸(DNTP)的存在使跨易位步骤的平衡向易位后状态移动。在这里，我们定量地确定了phi29 DNAP易位步骤与dNTP结合之间的动力学关系。我们证明了dNTP只有在从易位前状态转换到易位后状态后才能与phi29 DNAP-DNA复合体结合；dNTP结合纠正了易位，但它不直接驱动易位。根据测得的电流幅度的时间轨迹，我们发展了一种方法，分别在每种dNTP浓度和每种电压下确定正向和反向易位速率以及dNTP结合和解离速率。移位率及其对力的反应与phi29 DNAP-DNA二元复合体测定的结果一致，不受dNTP的影响。DNTP结合和解离速率不随电压变化，表明力不扭曲聚合酶活性部位，dNTP结合不直接涉及易位方向的移位。这种实验和理论相结合的方法以及所获得的结果，为分别评估生物变量对易位转变的影响及其对dNTP结合的影响提供了一个框架。
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.
登录
查看更多内容
影响因子：
15
作者：
Lieberman KR;Cherf GM;Doody MJ;Olasagasti F;Kolodji Y;Akeson M
通讯作者：
Akeson M
影响因子：
64.5
作者：
Franklin, MC;Wang, JM;Steitz, TA
通讯作者：
Steitz, TA
影响因子：
11.4
作者：
Berman, Andrea J.;Kamtekar, Satwik;Steitz, Thomas A.
通讯作者：
Steitz, Thomas A.
影响因子：
46.9
作者：
通讯作者：
--
影响因子：
4.8
作者：
Dahl, Joseph M.;Mai, Ai H.;Lieberman, Kate R.
通讯作者：
Lieberman, Kate R.