Zhang Jichuan, Zhou Ruobo, Inoue Jin, Mikawa Tsutomu, Ha Taekjip
Department of Physics and Center for the Physics of Living Cells, University of Illinois, Urbana, IL 61801, USA, Department of Materials Science and Engineering, University of Illinois, Urbana, IL 61801, USA, RIKEN, Cellular and Molecular Biology Unit, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan and Howard Hughes Medical Institute, University of Illinois, Urbana, IL 61801, USA.
Nucleic Acids Res. 2014 Apr;42(6):3821-32. doi: 10.1093/nar/gkt1316. Epub 2013 Dec 25.
Single-stranded (ss) DNA binding (SSB) proteins play central roles in DNA replication, recombination and repair in all organisms. We previously showed that Escherichia coli (Eco) SSB, a homotetrameric bacterial SSB, undergoes not only rapid ssDNA-binding mode transitions but also one-dimensional diffusion (or migration) while remaining bound to ssDNA. Whereas the majority of bacterial SSB family members function as homotetramers, dimeric SSB proteins were recently discovered in a distinct bacterial lineage of extremophiles, the Thermus-Deinococcus group. Here we show, using single-molecule fluorescence resonance energy transfer (FRET), that homodimeric bacterial SSB from Thermus thermophilus (Tth) is able to diffuse spontaneously along ssDNA over a wide range of salt concentrations (20-500 mM NaCl), and that TthSSB diffusion can help transiently melt the DNA hairpin structures. Furthermore, we show that two TthSSB molecules undergo transitions among different DNA-binding modes while remaining bound to ssDNA. Our results extend our previous observations on homotetrameric SSBs to homodimeric SSBs, indicating that the dynamic features may be shared among different types of SSB proteins. These dynamic features of SSBs may facilitate SSB redistribution and removal on/from ssDNA, and help recruit other SSB-interacting proteins onto ssDNA for subsequent DNA processing in DNA replication, recombination and repair.
单链（ss）DNA结合（SSB）蛋白在所有生物体的DNA复制、重组和修复过程中发挥着核心作用。我们之前发现，大肠杆菌（Eco）SSB，一种同四聚体细菌SSB，不仅会经历快速的ssDNA结合模式转变，还会在与ssDNA结合时发生一维扩散（或迁移）。虽然大多数细菌SSB家族成员以同四聚体形式发挥作用，但最近在嗜热栖热菌 - 嗜热放线菌属的一个独特细菌谱系中发现了二聚体SSB蛋白。在这里，我们使用单分子荧光共振能量转移（FRET）表明，嗜热栖热菌（Tth）的同二聚体细菌SSB能够在广泛的盐浓度范围（20 - 500 mM NaCl）内沿ssDNA自发扩散，并且TthSSB扩散有助于瞬时解开DNA发夹结构。此外，我们表明两个TthSSB分子在与ssDNA结合时会在不同的DNA结合模式之间转变。我们的结果将我们之前对同四聚体SSB的观察扩展到了同二聚体SSB，表明这些动态特征可能在不同类型的SSB蛋白中共享。SSB的这些动态特征可能有助于SSB在ssDNA上的重新分布和去除，并有助于将其他与SSB相互作用的蛋白招募到ssDNA上，以便在DNA复制、重组和修复过程中进行后续的DNA加工。
