Single-molecule optical mapping of the distribution of DNA phosphorothioate epigenetics.
Single-molecule optical mapping of the distribution of DNA phosphorothioate epigenetics.
复制标题
DNA 硫代磷酸酯表观遗传学分布的单分子光学图谱。
DOI：
10.1093/nar/gkab169
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发表时间：
2021-04-19
影响因子：
14.9
通讯作者：
Wang L
中科院分区：
文献类型：
--
作者：
Wei Y;Huang Q;Tian X;Zhang M;He J;Chen X;Chen C;Deng Z;Li Z;Chen S;Wang L
DNA硫代修饰是由DndABCDE或SspABCD控制的由硫取代非桥磷氧的修饰，广泛存在于原核生物中，具有一个非常不寻常的特征，即只占据基因组中可用共识序列的一小部分。尽管存在大量不受PT保护的共识，但DNA PT修饰仍然被限制同源基因(如DndFGH或SSPE)用作识别标签，以区分和破坏缺少PT的外源DNA。这提出了一个基本的问题，即PT修饰是如何沿着DNA分子分布的，以保持限制成分的受控。在这里，我们提出了两种单分子策略，这两种策略利用PT的亲核性与荧光标记相结合，用于光学映射单个DNA分子上的单链和双链PT修饰。令人惊讶的是，PT图谱因分子而异，即使在DndFGH或SSPE存在的情况下，PT位置和PT对之间的间距也不同。结果揭示了前所未有的PT修饰特征以前被集合平均所掩盖，为通过PT修饰和限制成分进行不寻常的靶点选择提供了新的见解。
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.
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