Demonstration of unidirectional single-stranded DNA translocation by PcrA helicase: Measurement of step size and translocation speed
Demonstration of unidirectional single-stranded DNA translocation by PcrA helicase: Measurement of step size and translocation speed
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DOI：
10.1021/bi992105o
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发表时间：
2000-01-11
期刊：
影响因子：
2.9
通讯作者：
Webb, MR
中科院分区：
文献类型：
--
作者：
Dillingham, MS;Wigley, DB;Webb, MR
使用无机磷酸盐的荧光传感器，在饱和浓度的各种长度的寡核苷酸的存在下，测量由PcrA解旋酶的ATP水解的动力学。有一个无机磷酸盐释放的快速阶段，相当于ATP酶的几次周转，然后是较慢的稳态ATP水解。快速相的大小由单链DNA的长度决定，而慢相与其长度无关。提出了一个动力学模型，其中快速相与易位沿着单链DNA，后的PcrA随机结合沿着DNA。单链DNA的长度与快速相的持续时间和振幅之间存在线性关系。这些数据表明，易位活动发生在50个碱基/秒的单向单碱基步骤，每个需要水解1个ATP分子。
Using a fluorescent sensor for inorganic phosphate, the kinetics of ATP hydrolysis by PcrA helicase were measured in the presence of saturating concentrations of oligonucleotides of various lengths. There is a rapid phase of inorganic phosphate release that is equivalent to several turnovers of the ATPase, followed by slower steady-state ATP hydrolysis. The magnitude of the rapid phase is governed by the length of single-stranded DNA, while the slow phase is independent of its length. A kinetic model is presented in which the rapid phase is associated with translocation along single-stranded DNA, after the PcrA binds randomly along the DNA. There is a linear relationship between the length of single-stranded DNA and both the duration and amplitude of the rapid phase. These data suggest that the translocation activity occurs at 50 bases/s in unidirectional single-base steps, each requiring the hydrolysis of 1 ATP molecule.