Ministry of Education Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China.
Taihe Hospital, Hubei University of Medicine, Shiyan 442000, Hubei, China.
Nucleic Acids Res. 2021 Apr 19;49(7):3672-3680. doi: 10.1093/nar/gkab169.
DNA phosphorothioate (PT) modifications, with the nonbridging phosphate oxygen replaced by sulfur, governed by DndABCDE or SspABCD, are widely distributed in prokaryotes and have a highly unusual feature of occupying only a small portion of available consensus sequences in a genome. Despite the presence of plentiful non-PT-protected consensuses, DNA PT modification is still employed as a recognition tag by the restriction cognate, for example, DndFGH or SspE, to discriminate and destroy PT-lacking foreign DNA. This raises a fundamental question about how PT modifications are distributed along DNA molecules to keep the restriction components in check. Here, we present two single-molecule strategies that take advantage of the nucleophilicity of PT in combination with fluorescent markers for optical mapping of both single- and double-stranded PT modifications across individual DNA molecules. Surprisingly, PT profiles vary markedly from molecule to molecule, with different PT locations and spacing distances between PT pairs, even in the presence of DndFGH or SspE. The results revealed unprecedented PT modification features previously obscured by ensemble averaging, providing novel insights into the riddles regarding unusual target selection by PT modification and restriction components.
DNA 硫代磷酸酯 (PT) 修饰，其中非桥接的磷酸氧被硫取代，由 DndABCDE 或 SspABCD 调控，广泛分布于原核生物中，具有一个非常不寻常的特征，即仅占据基因组中可用共有序列的一小部分。尽管存在大量非 PT 保护的共有序列，但 DNA PT 修饰仍然被限制酶的同源物用作识别标记，例如 DndFGH 或 SspE，以区分和破坏缺乏 PT 的外来 DNA。这就提出了一个基本问题，即 PT 修饰是如何沿着 DNA 分子分布的，以控制限制成分。在这里，我们提出了两种单分子策略，利用 PT 的亲核性，结合荧光标记，对单个 DNA 分子中双链和单链 PT 修饰进行光学作图。令人惊讶的是，PT 图谱在不同分子之间差异显著，PT 位置和 PT 对之间的间隔距离不同，即使存在 DndFGH 或 SspE 也是如此。这些结果揭示了以前被平均化掩盖的前所未有的 PT 修饰特征，为 PT 修饰和限制成分对不寻常靶标的选择之谜提供了新的见解。
