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首页> 外文期刊>Journal of Molecular Biology >Unzipping Mechanism of the Double-stranded DNA Unwinding by a Hexameric Helicase: Quantitative Analysis of the Rate of the dsDNA Unwinding, Processivity and Kinetic Step-size of the Escherichia coli DnaB Helicase Using Rapid Quench-flow Method.
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Unzipping Mechanism of the Double-stranded DNA Unwinding by a Hexameric Helicase: Quantitative Analysis of the Rate of the dsDNA Unwinding, Processivity and Kinetic Step-size of the Escherichia coli DnaB Helicase Using Rapid Quench-flow Method.

机译：六聚解旋酶解链双链DNA的解链机理：使用快速淬灭流方法对dsDNA解链速率，大肠杆菌DnaB解旋酶的动力学步长和动力学步幅进行定量分析。

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Kinetics of the double-stranded (ds) DNA unwinding by the Escherichia coli replicative helicase DnaB protein has been examined under single-turnover conditions using the chemical quench-flow technique. The unwinding reaction proceeds through an initial conformational transition followed by the unwinding catalytic steps and the release of the single-stranded (ss) DNA. Analyses of the reaction as a function of the number of base-pairs in the dsDNA reveal that the number of catalytic steps is not strictly proportional to the length of the dsDNA. As the helicase approaches the end of the substrate, the remaining approximately 11bp of the DNA melts without catalytic participation of the enzyme. The kinetic step-size of the DnaB helicase, i.e. the number of the base-pairs unwound in a single catalytic step is only 1.4(+/-0.2). The low value of the step-size indicates that the helicase unwinds a single base-pair in a single catalytic step. Thus, the DnaB helicase unzips the dsDNA in a reverse process to the zipping mechanism of the non-enzymatic double helix formation. The protein is a fast helicase that at 25 degrees C unwinds approximately 291bp/s, much faster than previously thought, and the unwinding rate can be much higher at higher temperatures. However, the ATP-state of the enzyme has an increased dissociation rate, resulting in only a moderate unwinding processivity, P=0.89(+/-0.03), little dependent on the temperature. The conformational transition of the DnaB helicase-DNA complex, preceding the unwinding, is an intrinsic transition of the enzyme from the stationary conformation to the ATP-state of the helicase.
机译：已使用化学淬灭流技术在单周转条件下检查了大肠杆菌复制性解旋酶DnaB蛋白解链的双链（ds）DNA的动力学。展开反应通过最初的构象转变进行，接着展开催化步骤并释放单链（ss）DNA。根据dsDNA中碱基对数目的函数对反应的分析表明，催化步骤的数目并不严格地与dsDNA的长度成正比。当解旋酶接近底物末端时，剩余的约11bp DNA融化而没有酶的催化参与。 DnaB解旋酶的动力学步长大小，即在单个催化步骤中解绕的碱基对的数目仅为1.4（+/- 0.2）。步长值低表明解旋酶在单个催化步骤中解开单个碱基对。因此，DnaB解旋酶以与非酶促双螺旋形成的拉链机制相反的过程将dsDNA解压缩。该蛋白是一种快速解旋酶，在25摄氏度下解旋速度约为291bp / s，比以前认为的快得多，在更高的温度下解旋速率可能更高。但是，该酶的ATP状态具有较高的解离速率，仅导致中等程度的解旋过程，P = 0.89（+/- 0.03），几乎不依赖于温度。在展开之前，DnaB解旋酶-DNA复合物的构象转变是酶从固定构象向解旋酶的ATP状态的固有转变。

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