Direct Observation of Translocation in Individual DNA Polymerase Complexes
Direct Observation of Translocation in Individual DNA Polymerase Complexes
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DOI：
10.1074/jbc.m111.338418
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发表时间：
2012-04-13
影响因子：
4.8
通讯作者：
Lieberman, Kate R.
中科院分区：
文献类型：
--
作者：
Dahl, Joseph M.;Mai, Ai H.;Lieberman, Kate R.
phi 29 DNA聚合酶和DNA的复合物在施加的电场中在α-溶血素纳米孔顶部捕获时在两个离子电流振幅状态之间以毫秒时间尺度波动。较低振幅状态通过互补dNTP稳定，因此对应于易位后状态的复合物。我们已经证明，在上振幅状态下，DNA从易位后状态移位一个核苷酸的距离。我们建议，上振幅状态对应于复合物的前易位状态。施加在模板链上的力使复合物偏向易位前状态。基于电压和dNTP滴定的结果，我们通过数学建模得出结论，互补dNTP仅结合到易位后状态，并且我们估计了结合亲和力。这两种状态之间的平衡受到活性位点附近的DNA序列的影响。与将上振幅状态指定为预易位状态一致，有利于纳米孔上复合物中的预易位状态的DNA底物是体相中用于焦磷酸解的上级底物。也有一个相关性的DNA序列，偏向复合物的易位前状态和外切核酸溶解率在本体阶段，这表明在DNA合成的引物链从聚合酶转移到核酸外切酶活性位点的途径开始在易位前状态。
Complexes of phi29 DNA polymerase and DNA fluctuate on the millisecond time scale between two ionic current amplitude states when captured atop the alpha-hemolysin nanopore in an applied field. The lower amplitude state is stabilized by complementary dNTP and thus corresponds to complexes in the post-translocation state. We have demonstrated that in the upper amplitude state, the DNA is displaced by a distance of one nucleotide from the post-translocation state. We propose that the upper amplitude state corresponds to complexes in the pre-translocation state. Force exerted on the template strand biases the complexes toward the pre-translocation state. Based on the results of voltage and dNTP titrations, we concluded through mathematical modeling that complementary dNTP binds only to the post-translocation state, and we estimated the binding affinity. The equilibrium between the two states is influenced by active site-proximal DNA sequences. Consistent with the assignment of the upper amplitude state as the pre-translocation state, a DNA substrate that favors the pre-translocation state in complexes on the nanopore is a superior substrate in bulk phase for pyrophosphorolysis. There is also a correlation between DNA sequences that bias complexes toward the pre-translocation state and the rate of exonucleolysis in bulk phase, suggesting that during DNA synthesis the pathway for transfer of the primer strand from the polymerase to exonuclease active site initiates in the pre-translocation state.