Single molecule measurements of DNA helicase activity with magnetic tweezers and t-test based step-finding analysis.
Single molecule measurements of DNA helicase activity with magnetic tweezers and t-test based step-finding analysis.
复制标题
用磁性镊子和基于t检验的台阶分析对DNA解旋酶活性的单分子测量。
DOI：
10.1016/j.ymeth.2016.04.030
复制
发表时间：
2016-08-01
期刊：
影响因子：
--
通讯作者：
Neuman KC
中科院分区：
文献类型：
--
作者：
Seol Y;Strub MP;Neuman KC
磁镊是一种多功能且易于实现的单分子技术，在基于核酸的分子马达的研究中变得越来越普遍。在这里，我们提供了一个磁镊子仪器和测量和分析DNA解旋酶活性的指导方针的描述。沿着实验方法，我们描述了一个强大的方法，单分子轨迹分析的基础上，学生的t-检验，除了最广泛采用的分析程序中假设的离散过渡，可容纳连续过渡。为了说明单分子解旋测定和分析程序，我们提供了在各种条件下（Na+，ATP，温度和DNA底物几何形状）的大肠杆菌RecQ解旋酶的DNA解旋测量。这些实施例表明，在各种条件下的DNA解旋测量可以帮助阐明DNA解旋酶的解旋机制，但也强调，环境对DNA解旋酶活性的影响，必须考虑在体内的活动和机制。
Magnetic tweezers is a versatile and easy to implement single-molecule technique that has become increasingly prevalent in the study of nucleic acid based molecular motors. Here, we provide a description of the magnetic tweezers instrument and guidelines for measuring and analyzing DNA helicase activity. Along with experimental methods, we describe a robust method of single-molecule trajectory analysis based on the Student’s t-test that accommodates continuous transitions in addition to the discrete transitions assumed in most widely employed analysis routines. To illustrate the single-molecule unwinding assay and the analysis routine, we provide DNA unwinding measurements of Escherichia coli RecQ helicase under a variety of conditions (Na+, ATP, temperature, and DNA substrate geometry). These examples reveal that DNA unwinding measurements under various conditions can aid in elucidating the unwinding mechanism of DNA helicase but also emphasize that environmental effects on DNA helicase activity must be considered in relation to in vivo activity and mechanism.