MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
Methods. 2010 Jul;51(3):259-68. doi: 10.1016/j.ymeth.2010.02.012. Epub 2010 Feb 16.
Methods are described to show how different fluorescent labeling strategies can be used to probe various aspects of the helicase mechanism. Fluorophores on the adenine nucleotide, the DNA or the helicase can modify the activity of the system to a greater or lesser extent. Reagentless biosensors, binding proteins that are labeled with a fluorophore, target products of the helicase reaction, namely ADP, inorganic phosphate or single-stranded DNA, and can be used to measure rates of product formation with little interference to the system. Protocols are described to examine ATP usage and translocation speeds and also to investigate details of the ATP hydrolysis cycle. The methods are described in terms of PcrA, a bacterial DNA helicase that moves in single base steps along either single-stranded or double-stranded DNA, hydrolyzing one ATP per base moved.
方法描述了如何使用不同的荧光标记策略来探测解旋酶机制的各个方面。腺嘌呤核苷酸、DNA 或解旋酶上的荧光团可以在不同程度上修饰系统的活性。无试剂生物传感器，即标记有荧光团的结合蛋白，可以靶向解旋酶反应的产物，即 ADP、无机磷酸或单链 DNA，并可用于测量产物形成的速率，而对系统的干扰很小。本文描述了检测 ATP 使用和转运速度的方案，以及研究 ATP 水解循环细节的方案。这些方法是针对 PcrA 描述的，PcrA 是一种细菌 DNA 解旋酶，可以沿着单链或双链 DNA 以单碱基步长移动，每移动一个碱基水解一个 ATP。
