Song Dan, Rodrigues Kristen, Graham Thomas G W, Loparo Joseph J
Harvard Biophysics Program, Harvard Medical School, Boston MA 02115, USA.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Nucleic Acids Res. 2017 Jul 7;45(12):7106-7117. doi: 10.1093/nar/gkx271.
Most bacteria utilize the highly conserved parABS partitioning system in plasmid and chromosome segregation. This system depends on a DNA-binding protein ParB, which binds specifically to the centromere DNA sequence parS and to adjacent non-specific DNA over multiple kilobases in a phenomenon called spreading. Previous single-molecule experiments in combination with genetic, biochemical and computational studies have argued that ParB spreading requires cooperative interactions between ParB dimers including DNA bridging and possible nearest-neighbor interactions. A recent structure of a ParB homolog co-crystallized with parS revealed that ParB dimers tetramerize to form a higher order nucleoprotein complex. Using this structure as a guide, we systematically ablated a series of proposed intermolecular interactions in the Bacillus subtilis ParB (BsSpo0J) and characterized their effect on spreading using both in vivo and in vitro assays. In particular, we measured DNA compaction mediated by BsSpo0J using a recently developed single-molecule method to simultaneously visualize protein binding on single DNA molecules and changes in DNA conformation without protein labeling. Our results indicate that residues acting as hubs for multiple interactions frequently led to the most severe spreading defects when mutated, and that a network of both cis and trans interactions between ParB dimers is necessary for spreading.
大多数细菌在质粒和染色体分离过程中利用高度保守的parABS分区系统。该系统依赖于一种DNA结合蛋白ParB，它特异性地结合着丝粒DNA序列parS，并在数千碱基的范围内与相邻的非特异性DNA结合，这种现象称为扩散。先前的单分子实验结合遗传、生化和计算研究表明，ParB扩散需要ParB二聚体之间的协同相互作用，包括DNA桥接和可能的近邻相互作用。最近一个与parS共结晶的ParB同源物的结构显示，ParB二聚体四聚化形成更高阶的核蛋白复合物。以这个结构为指导，我们系统地消除了枯草芽孢杆菌ParB（BsSpo0J）中一系列提出的分子间相互作用，并使用体内和体外试验来表征它们对扩散的影响。特别是，我们使用最近开发的单分子方法测量了BsSpo0J介导的DNA压缩，该方法可以在不进行蛋白质标记的情况下同时可视化单个DNA分子上的蛋白质结合和DNA构象变化。我们的结果表明，作为多种相互作用枢纽的残基在突变时经常导致最严重的扩散缺陷，并且ParB二聚体之间的顺式和反式相互作用网络对于扩散是必要的。
