Precise sequencing of single protected-DNA fragment molecules for profiling of protein distribution and assembly on DNA,Chemical Science

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Precise sequencing of single protected-DNA fragment molecules for profiling of protein distribution and assembly on DNA
Chemical Science ( IF 7.6 ) Pub Date : 2021-1-2 , DOI: 10.1039/d0sc01742f
Zheng Yuan _{1,

2} , Dapeng Zhang _{1,

3} , Fangzhi Yu _{1,

2} , Yangde Ma ₂ , Yan Liu _{1,

2} , Xiangjun Li ₂ , Hailin Wang _{1,

2,

3,

4}

Affiliation

Multiple DNA-interacting protein molecules are often dynamically distributed and/or assembled along a DNA molecule to adapt to their intricate functions temporally. However, analytical technology for measuring such binding behaviours is still missing. Here, we demonstrate the unique capacity of a supernuclease for a highly efficient cutting of the unprotected-DNA segments and with complete preservation of the protein-occluded DNA segments at near single-nucleotide resolution. By exploring this high-resolution cutting, an unprecedented assay that allows a precise sequencing of single protected-DNA fragment molecules (SPDFMS) was developed. As relevant applications, relevant information was gained on the respective distribution/assembly patterns and coordinated displacement of single-stranded DNA-binding protein and recombinase RecA, two model proteins, on DNA. Benefiting from this assay, we also for the first time provide direct measurement of the length of single RecA nucleofilaments, showing the predominant stoichiometry of 5–7 RecA monomers per RecA nucleofilament under physiologically relevant conditions. This innovative assay appears as a promising analytical tool for studying diverse protein–DNA interactions implicated in DNA replication, transcription, recombination, repair, and gene editing.

中文翻译：

对单个受保护 DNA 片段分子进行精确测序，以分析 DNA 上的蛋白质分布和组装情况

多个 DNA 相互作用蛋白分子通常沿着 DNA 分子动态分布和/或组装，以暂时适应其复杂的功能。然而，仍然缺乏测量这种结合行为的分析技术。在这里，我们展示了超级核酸酶的独特能力，可以高效切割未受保护的 DNA 片段，并以接近单核苷酸的分辨率完全保存蛋白质封闭的 DNA 片段。通过探索这种高分辨率切割，开发了一种前所未有的检测方法，可以对单个受保护 DNA 片段分子 (SPDFMS) 进行精确测序。作为相关应用，获得了两种模型蛋白单链DNA结合蛋白和重组酶RecA各自在DNA上的分布/组装模式和协调位移的相关信息。受益于该测定，我们还首次提供了单个 RecA 核丝长度的直接测量，显示了在生理相关条件下每个 RecA 核丝 5-7 个 RecA 单体的主要化学计量。这种创新的检测方法似乎是一种很有前景的分析工具，可用于研究 DNA 复制、转录、重组、修复和基因编辑中涉及的多种蛋白质-DNA 相互作用。

更新日期：2021-01-27

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