Laboratoire de Stress Génotoxiques et Cancer, CNRS UMR3348, Institut Curie-Section de Recherche, Centre Universitaire, Bat 110, 91405 Orsay, France.
Methods. 2010 Jul;51(3):289-94. doi: 10.1016/j.ymeth.2010.02.022. Epub 2010 Feb 26.
In order to elucidate molecular mechanism of helicases, we have developed two new rapid and sensitive fluorescence assays to measure helicase-mediated DNA unwinding. The fluorescence anisotropy (FA) assay takes the advantage of the substantial change in fluorescence polarization upon helicase binding to DNA and DNA unwinding. The extent of depolarization depends on the rate of tumbling of the fluorescently labeled DNA molecule, which decreases with increasing size. This assay therefore can simultaneously monitor the DNA binding of helicase and the subsequent helicase-catalyzed DNA unwinding in real-time. For size limitation reasons, the FA approach is more suitable for single-turnover kinetic studies. A fluorescence cross-correlation spectroscopy method (FCCS) is also described for measuring DNA unwinding. This assay is based on the degree of concomitant diffusion of the two complementary DNA strands in a small excitation volume, each labeled by a different color. The decrease in the amplitude of the cross-correlation signal is then directly related to the unwinding activity. By contrast with FA, the FCCS-based assay can be used to measure the unwinding activity under both single- and multiple-turnover conditions, with no limitation related to the size of the DNA strands constituting the DNA substrate. These methods used together have proven to be useful for studying molecular mechanism underlying efficient motor function of helicases. Here, we describe the theoretical basis and framework of FA and FCCS and some practical implications for measuring DNA binding and unwinding. We discuss sample preparation and potential troubleshooting. Special attention is paid to instrumentation, data acquisition and analysis.
为了阐明解旋酶的分子机制，我们开发了两种新的快速灵敏的荧光测定法来测量解旋酶介导的 DNA 解旋。荧光各向异性（FA）测定法利用解旋酶与 DNA 结合和 DNA 解旋时荧光偏振的显著变化。去极化的程度取决于荧光标记的 DNA 分子的旋转程度，随着分子大小的增加而降低。因此，该测定法可以实时监测解旋酶的 DNA 结合及其随后的解旋酶催化的 DNA 解旋。由于尺寸限制的原因，FA 方法更适合单轮动力学研究。还描述了一种荧光相关光谱法（FCCS）来测量 DNA 解旋。该测定法基于在小激发体积中两个互补 DNA 链伴随扩散的程度，每个链用不同的颜色标记。然后，互相关信号的幅度减小与解旋活性直接相关。与 FA 相比，基于 FCCS 的测定法可用于测量单轮和多轮条件下的解旋活性，不受构成 DNA 底物的 DNA 链大小的限制。这些方法一起使用已被证明对研究解旋酶高效运动功能的分子机制很有用。在这里，我们描述了 FA 和 FCCS 的理论基础和框架，以及测量 DNA 结合和解旋的一些实际意义。我们讨论了样品制备和潜在的故障排除。特别注意仪器，数据采集和分析。
