Specific nucleotide binding and rebinding to individual DNA polymerase complexes captured on a nanopore.
Specific nucleotide binding and rebinding to individual DNA polymerase complexes captured on a nanopore.
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DOI：
10.1021/ja809663f
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发表时间：
2009-03-18
影响因子：
15
通讯作者：
Lieberman KR
中科院分区：
文献类型：
--
作者：
Hurt N;Wang H;Akeson M;Lieberman KR
纳米孔是DNA或RNA加工酶单分子分析的工具。使用这种技术实时监测催化活性需要这些酶在外加电场中保持在纳米孔上的功能。利用α-溶血素纳米孔，我们测量了DNA与大肠杆菌DNA聚合酶I （KF）的Klenow片段复合物的停留时间与脱氧核苷三磷酸（dNTP）底物浓度的关系。我们在捕获复合物（DNA-KF二元态和DNA-KF- dntp三元态）的双态模型框架下分析了这些停留时间测量。平均纳米孔停留时间增加而不饱和，作为正确dNTP浓度的函数跨越四个数量级。这源于与dNTP浓度成正比的两个因素：1)最初捕获时处于三元态的配合物的比例主要影响低dNTP浓度下的停留时间；2)在较高的dNTP浓度下，dNTP与捕获复合物的结合和再结合速率影响停留时间。因此，在平均停留时间和dNTP浓度之间存在两种显示线性关系的制度。从一种线性状态到另一种线性状态的转变发生在溶液中dNTP与KF-DNA复合物结合的平衡解离常数（Kd）附近。我们从滴定实验和建模的结合中得出结论，纳米孔顶部捕获的DNA- kf复合物保留了迭代的、序列特异性的dNTP结合，这是DNA合成催化和保真度所必需的。
Nanoscale pores are a tool for single molecule analysis of DNA or RNA processing enzymes. Monitoring catalytic activity in real time using this technique requires that these enzymes retain function while held atop a nanopore in an applied electric field. Using an α-hemolysin nanopore, we measured the dwell time for complexes of DNA with the Klenow fragment of Escherichia coli DNA polymerase I (KF) as a function of the concentration of deoxynucleoside triphosphate (dNTP) substrate. We analyzed these dwell time measurements in the framework of a two-state model for captured complexes (DNA-KF binary and DNA-KF-dNTP ternary states). Average nanopore dwell time increased without saturating as a function of correct dNTP concentration across four orders of magnitude. This arises from two factors that are proportional to dNTP concentration: 1) The fraction of complexes that are in the ternary state when initially captured predominantly affects dwell time at low dNTP concentrations; 2) The rate of binding and rebinding of dNTP to captured complexes affects dwell time at higher dNTP concentrations. Thus there are two regimes that display a linear relationship between average dwell time and dNTP concentration. The transition from one linear regime to the other occurs near the equilibrium dissociation constant (Kd) for dNTP binding to KF-DNA complexes in solution. We conclude from the combination of titration experiments and modeling that DNA-KF complexes captured atop the nanopore retain iterative, sequence-specific dNTP binding, as required for catalysis and fidelity in DNA synthesis.
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影响因子：
15
作者：
Cockroft, Scott L.;Chu, John;Ghadiri, M. Reza
通讯作者：
Ghadiri, M. Reza
影响因子：
2.9
作者：
Joyce, Catherine M.;Potapova, Olga;Grindley, Nigel D. F.
通讯作者：
Grindley, Nigel D. F.
影响因子：
8
作者：
Li, Y;Kong, Y;Waksman, G
通讯作者：
Waksman, G
DOI：
10.1073/pnas.93.24.13770
发表时间：
1996-11-26
影响因子：
11.1
作者：
Kasianowicz, JJ;Brandin, E;Deamer, DW
通讯作者：
Deamer, DW
影响因子：
2.9
作者：
KUCHTA, RD;MIZRAHI, V;BENKOVIC, SJ
通讯作者：
BENKOVIC, SJ