Dynamics of the translocation step measured in individual DNA polymerase complexes.
Dynamics of the translocation step measured in individual DNA polymerase complexes.
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DOI：
10.1021/ja3090302
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发表时间：
2012-11-14
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.;Akeson, Mark;Wang, Hongyun
噬菌体phi 29 DNA聚合酶（DNAP）和DNA之间形成的复合物在毫秒时间尺度上在易位前和易位后状态之间波动。当在施加的电场中在α-溶血素纳米孔顶部捕获单个复合物时，可以在实时离子电流迹线中以单核苷酸精度直接观察到这些波动。我们最近量化了易位步骤的平衡，作为施加的力（电压），活性位点近端DNA序列和互补dNTP的结合的函数。为了深入了解DNAP催化循环中这一步骤的机制，在这项研究中，我们研究了易位步骤的随机动力学。复合物在这两种状态下的存活概率都以单一的指数速率衰减，这表明所观察到的波动是在两个离散状态之间。我们使用基于自相关函数的稳健数学公式从捕获的phi 29 DNAP-DNA二元复合物的离子电流迹线提取易位前状态和易位后状态之间的转变的正向和反向速率。我们评估了作为施加电压的函数的每个转变速率，以检查phi 29 DNAP易位步骤的能量景观。分析表明，活性位点附近的DNA序列的影响的深度的前易位和易位后的状态能量威尔斯和影响的位置的过渡态沿着的方向的易位。
Complexes formed between the bacteriophage phi29 DNA polymerase (DNAP) and DNA fluctuate between the pre-translocation and post-translocation states on the millisecond time scale. These fluctuations can be directly observed with single-nucleotide precision in real-time ionic current traces when individual complexes are captured atop the alpha-hemolysin nanopore in an applied electric field. We recently quantified the equilibrium across the translocation step as a function of applied force (voltage), active-site proximal DNA sequences, and the binding of complementary dNTP. To gain insight into the mechanism of this step in the DNAP catalytic cycle, in this study, we have examined the stochastic dynamics of the translocation step. The survival probability of complexes in each of the two states decayed at a single exponential rate, indicating that the observed fluctuations are between two discrete states. We used a robust mathematical formulation based on the auto-correlation function to extract the forward and reverse rates of the transitions between the pre-translocation state and the post-translocation state from ionic current traces of captured phi29 DNAP-DNA binary complexes. We evaluated each transition rate as a function of applied voltage to examine the energy landscape of the phi29 DNAP translocation step. The analysis reveals that active-site proximal DNA sequences influence the depth of the pre-translocation and post-translocation state energy wells and affect the location of the transition state along the direction of the translocation.
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影响因子：
15
作者：
Hurt N;Wang H;Akeson M;Lieberman KR
通讯作者：
Lieberman KR
影响因子：
4.8
作者：
Wang, Weina;Wu, Eugene Y.;Beese, Lorena S.
通讯作者：
Beese, Lorena S.
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.