Institute of Molecular Genetics, NRC "Kurchatov Institute", Moscow, 123182, Russia.
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125, USA.
Biochem Biophys Res Commun. 2020 Dec 17;533(4):1484-1489. doi: 10.1016/j.bbrc.2020.10.051. Epub 2020 Oct 24.
In contrast to eukaryotic Argonaute proteins that act on RNA targets, prokaryotic Argonautes (pAgos) can target DNA, using either small RNA or small DNA guides for its recognition. Since pAgos can recognize only a single strand of DNA and lack a helicase activity, it remains unknown how double-stranded DNA can be bound both in vitro and in vivo. Here, using in vitro reconstitution and footprinting assays we analyze formation of specific complexes with target DNA by a catalytically inactive pAgo, RsAgo from Rhodobacter sphaeroides programmed with small guide RNAs. We showed that RsAgo can recognize a specific site in double-stranded DNA after stepwise reconstitution of the complex from individual oligonucleotides or after prior melting of the DNA target. When bound, RsAgo stabilizes an open DNA bubble corresponding to the length of the guide molecule and protects the target DNA from nuclease cleavage. The results suggest that RsAgo and, possibly, other RNA-guided pAgos cannot directly attack double-stranded DNA and likely require DNA opening by other cellular processes for their action.
与作用于 RNA 靶标的真核 Argonaute 蛋白不同，原核 Argonautes（pAgos）可以使用小 RNA 或小 DNA 向导来识别 DNA 靶标。由于 pAgos 只能识别 DNA 的单链，并且缺乏解旋酶活性，因此仍然不清楚双链 DNA 如何在体外和体内都能被结合。在这里，我们使用体外重组和足迹分析实验，分析了具有催化活性的 pAgo（来自 Rhodobacter sphaeroides 的 RsAgo）与小引导 RNA 编程的靶 DNA 形成特定复合物的过程。我们表明，RsAgo 可以在从单个寡核苷酸逐步重组复合物或在 DNA 靶标预先融化之后，识别双链 DNA 中的特定位点。当结合时，RsAgo 稳定与引导分子长度相对应的开放 DNA 泡，并保护靶 DNA 免受核酸酶切割。结果表明，RsAgo 可能还有其他 RNA 指导的 pAgos 不能直接攻击双链 DNA，并且可能需要其他细胞过程来打开 DNA，以便它们发挥作用。
