MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.
Nucleic Acids Res. 2010 Jul;38(13):4448-57. doi: 10.1093/nar/gkq173. Epub 2010 Mar 28.
DNA helicases are motor proteins that catalyze the unwinding of double-stranded DNA into single-stranded DNA using the free energy from ATP hydrolysis. Single molecule approaches enable us to address detailed mechanistic questions about how such enzymes move processively along DNA. Here, an optical method has been developed to follow the unwinding of multiple DNA molecules simultaneously in real time. This was achieved by measuring the accumulation of fluorescent single-stranded DNA-binding protein on the single-stranded DNA product of the helicase, using total internal reflection fluorescence microscopy. By immobilizing either the DNA or helicase, localized increase in fluorescence provides information about the rate of unwinding and the processivity of individual enzymes. In addition, it reveals details of the unwinding process, such as pauses and bursts of activity. The generic and versatile nature of the assay makes it applicable to a variety of DNA helicases and DNA templates. The method is an important addition to the single-molecule toolbox available for studying DNA processing enzymes.
DNA 解旋酶是一类利用 ATP 水解产生的自由能促使双链 DNA 解旋为单链 DNA 的分子马达。单分子技术使我们能够深入研究这些酶如何沿 DNA 进行连续运动的详细机制问题。在这里，我们开发了一种光学方法，能够实时同时跟踪多个 DNA 分子的解旋过程。该方法通过使用全内反射荧光显微镜测量荧光单链 DNA 结合蛋白在解旋酶产生的单链 DNA 产物上的积累来实现。通过固定 DNA 或解旋酶，可以通过局部荧光增加来提供有关单个酶解旋速度和连续性的信息。此外，它还揭示了解旋过程的细节，例如活性的暂停和爆发。该测定方法具有通用性和多功能性，适用于各种 DNA 解旋酶和 DNA 模板。该方法是研究 DNA 加工酶的单分子工具包的重要补充。
