A nonuniform stepping mechanism for E-coli UvrD monomer translocation along single-stranded DNA
A nonuniform stepping mechanism for E-coli UvrD monomer translocation along single-stranded DNA
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DOI：
10.1016/j.molcel.2007.03.024
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发表时间：
2007-05-11
期刊：
影响因子：
16
通讯作者：
Lohman, Timothy M.
中科院分区：
文献类型：
--
作者：
Tomko, Eric J.;Fischer, Christopher J.;Lohman, Timothy M.
E.coliUvrD是一种参与多种DNA代谢过程的SF1解旋酶。虽然解旋酶的活性需要UvrD晚餐，但单体可以沿着单链DNA以3‘到5’的方向移位，这种依赖于ATP的移位可能参与了RecA的移位。为了了解单体转位酶的功能，我们结合了荧光停流动力学方法和最近发展起来的分析方法来确定UvrD单体转位的动力学机制，包括ATP偶联化学计量学。我们的结果表明，UvrD单体转位的宏观速度不受每个ATPase周期的限制，而是受每个转位周期中的一个缓慢步骤(暂停)的限制，该步骤发生在4到5个快速的1个核苷酸转位步骤之后，每个快速步骤都与一个ATP的水解相耦合。这些结果表明了一种不同于布朗马达或以前基于结构的尺寸虫机构的非均匀步进机构。
E. coli UvrD is an SF1 helicase involved in several DNA metabolic processes. Although a UvrD dinner is needed for helicase activity, a monomer can translocate with 3' to 5' directionality along single-stranded DNA, and this ATP-dependent translocation is likely involved in RecA displacement. In order to understand how the monomeric translocase functions, we have combined fluorescence stopped-flow kinetic methods with recently developed analysis methods to determine the kinetic mechanism, including ATP coupling stoichiometry, for UvrD monomer translocation along ssDNA. Our results suggest that the macroscopic rate of UvrD monomer translocation is not limited by each ATPase cycle but rather by a slow step (pause) in each translocation cycle that occurs after four to five rapid 1 nt translocation steps, with each rapid step coupled to hydrolysis of one ATP. These results suggest a non-uniform stepping mechanism that differs from either a Brownian motor or previous structure-based inchworm mechanisms.