Zhou Ruobo, Ha Taekjip
Department of Physics and Center for the Physics of Living Cells, University of Illinois, Urbana-Champaign, IL, USA.
Methods Mol Biol. 2012;922:85-100. doi: 10.1007/978-1-62703-032-8_5.
SSB proteins bind to and control the accessibility of single-stranded (ss) DNA generated as a transient intermediate during a variety of cellular processes. For subsequent DNA processing, however, SSB needs to be removed and yield to other proteins while avoiding ssDNA exposure to nucleases. Using single-molecule two- and three-color fluorescence resonance energy transfer (FRET) and fluorescence-force spectroscopy, we recently showed that the SSB/DNA complex is a highly dynamic system and SSB functions as a sliding platform that migrates on ssDNA for recruiting other proteins in DNA repair, replication, and recombination. Here, we present the activity assays in detail for observing the transitions between different SSB binding modes and SSB diffusion on ssDNA in real time by using single-molecule FRET microscopy and for studying how mechanical forces regulate SSB-DNA interactions using fluorescence-force spectroscopy. These single-molecule approaches are generally applicable to many other protein-nucleic acid systems.
单链结合蛋白（SSB）可结合并控制在多种细胞过程中作为瞬时中间体产生的单链（ss）DNA的可及性。然而，为了后续的DNA加工，SSB需要被移除并让位于其他蛋白质，同时避免单链DNA暴露于核酸酶。我们最近利用单分子双色和三色荧光共振能量转移（FRET）以及荧光力谱表明，SSB/DNA复合物是一个高度动态的系统，并且SSB作为一个滑动平台在单链DNA上迁移，以在DNA修复、复制和重组过程中招募其他蛋白质。在这里，我们详细介绍了活性测定方法，通过使用单分子FRET显微镜实时观察不同SSB结合模式之间的转变以及SSB在单链DNA上的扩散，并利用荧光力谱研究机械力如何调节SSB-DNA相互作用。这些单分子方法通常适用于许多其他蛋白质-核酸系统。
