B CUBE - Center for Molecular Bioengineering, TU Dresden, Tatzberg 41, 01307 Dresden, Germany.
B CUBE - Center for Molecular Bioengineering, TU Dresden, Tatzberg 41, 01307 Dresden, Germany.
Methods. 2019 Oct 1;169:11-20. doi: 10.1016/j.ymeth.2019.02.011. Epub 2019 Feb 15.
Apart from being storage devices for genetic information, nucleic acids can provide regulatory structures through evolutionarily optimized sequences. The interaction of proteins binding specifically to such sequences and resulting secondary structures, or the exposure of single-stranded DNA add a versatile regulatory framework for cells. Biochemical and structural biology experiments have revealed important underlying concepts of protein-DNA interactions but are often limited by ensemble averaging or static information. To decipher the dynamics of conformations adopted by protein-DNA complexes, single-molecule approaches have become a powerful resource over the past two decades. In particular single-molecule FRET (smFRET), which allows a read-out of DNA or protein conformations, became widely used. Here, we illustrate how to implement the technique and exemplarily describe how smFRET yields insights into conformational changes of DNA secondary structures induced by the single-stranded DNA binding protein SSB. We further explain how we use smFRET to study mechanisms of the replication initiator DnaA and the competition of DnaA and SSB for single-stranded DNA. We anticipate that smFRET will further develop into a particularly useful technique to study dynamic competitions of proteins for the same DNA substrate.
除了作为遗传信息的存储设备外，核酸还可以通过进化优化的序列提供调节结构。蛋白质特异性结合这些序列并形成二级结构的相互作用，或单链 DNA 的暴露为细胞增加了一个多功能的调节框架。生化和结构生物学实验揭示了蛋白质-DNA 相互作用的重要基本概念，但往往受到整体平均或静态信息的限制。为了解密蛋白质-DNA 复合物所采用的构象动力学，单分子方法在过去二十年中已成为一种强大的资源。特别是单分子荧光共振能量转移（smFRET），它可以读取 DNA 或蛋白质构象，已被广泛应用。在这里，我们说明了如何实施该技术，并举例说明了 smFRET 如何深入了解单链结合蛋白 SSB 诱导的 DNA 二级结构构象变化。我们进一步解释了如何使用 smFRET 来研究复制起始蛋白 DnaA 的机制以及 DnaA 和 SSB 对单链 DNA 的竞争。我们预计 smFRET 将进一步发展成为一种特别有用的技术，用于研究蛋白质对同一 DNA 底物的动态竞争。
