Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.
Department of Physics and Astronomy, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.
Sci Rep. 2021 Sep 16;11(1):18492. doi: 10.1038/s41598-021-97763-w.
Architectural DNA-binding proteins are involved in many important DNA transactions by virtue of their ability to change DNA conformation. Histone-like protein from E. coli strain U93, HU, is one of the most studied bacterial architectural DNA-binding proteins. Nevertheless, there is still a limited understanding of how the interactions between HU and DNA are affected by ionic conditions and the structure of DNA. Here, using optical tweezers in combination with fluorescent confocal imaging, we investigated how ionic conditions affect the interaction between HU and DNA. We directly visualized the binding and the diffusion of fluorescently labelled HU dimers on DNA. HU binds with high affinity and exhibits low mobility on the DNA in the absence of Mg; it moves 30-times faster and stays shorter on the DNA with 8 mM Mg in solution. Additionally, we investigated the effect of DNA tension on HU-DNA complexes. On the one hand, our studies show that binding of HU enhances DNA helix stability. On the other hand, we note that the binding affinity of HU for DNA in the presence of Mg increases at tensions above 50 pN, which we attribute to force-induced structural changes in the DNA. The observation that HU diffuses faster along DNA in presence of Mg compared to without Mg suggests that the free energy barrier for rotational diffusion along DNA is reduced, which can be interpreted in terms of reduced electrostatic interaction between HU and DNA, possibly coinciding with reduced DNA bending.
结构 DNA 结合蛋白通过改变 DNA 构象来参与许多重要的 DNA 转化。来自大肠杆菌 U93 株的组蛋白样蛋白 HU 是研究最多的细菌结构 DNA 结合蛋白之一。尽管如此，人们对于 HU 与 DNA 之间的相互作用如何受到离子条件和 DNA 结构的影响仍然知之甚少。在这里，我们使用光学镊子结合荧光共焦成像技术，研究了离子条件如何影响 HU 与 DNA 之间的相互作用。我们直接可视化了荧光标记的 HU 二聚体在 DNA 上的结合和扩散。在没有 Mg 的情况下，HU 与 DNA 具有高亲和力且表现出低迁移率；在溶液中有 8 mM Mg 的情况下，它的移动速度快 30 倍，在 DNA 上停留的时间更短。此外，我们研究了 DNA 张力对 HU-DNA 复合物的影响。一方面，我们的研究表明，HU 的结合增强了 DNA 螺旋的稳定性。另一方面，我们注意到，在张力高于 50 pN 的情况下，Mg 存在时 HU 对 DNA 的结合亲和力增加，我们将其归因于 DNA 结构中力诱导的变化。与没有 Mg 相比，在 Mg 存在的情况下 HU 沿 DNA 扩散更快的观察结果表明，沿 DNA 进行旋转扩散的自由能障碍降低，这可以解释为 HU 与 DNA 之间的静电相互作用降低，可能与 DNA 弯曲减少有关。
