State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, Hubei 430062, China.
State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, Hubei 430062, China.
Cell Rep. 2022 Oct 25;41(4):111533. doi: 10.1016/j.celrep.2022.111533.
Argonaute proteins are widespread in prokaryotes and eukaryotes with diversified catalytic activities. Here, we describe an Argonaute from Marinitoga hydrogenitolerans (MhAgo) with all eight cleavage activities. Utilization of all four types of guides and efficient cleavage of single-stranded DNA (ssDNA) and RNA targets are revealed. The preference for the 5'-terminus nucleotides of 5'P guides, but no obvious preferences for that in 5'OH guides, is further uncovered. Moreover, the cleavage efficiency is heavily impaired by mismatches in the central and 3'-supplementary regions of guides, and the affinity between guides or guides/target duplex and MhAgo is proved as one of the factors affecting cleavage efficiency. Structural and mutational analyses imply some unknown distinctive structural features behind the cleavage activity of MhAgo. Meanwhile, 5'OH-guide RNA (gRNA)-mediated plasmid cleavage activity is unveiled. Conclusively, MhAgo is versatile, and its biochemical characteristics improve our understanding of pAgos and the pAgo-based techniques.
Argonaute 蛋白广泛存在于原核生物和真核生物中，具有多样化的催化活性。在这里，我们描述了来自 Marinitoga hydrogenitolerans（MhAgo）的 Argonaute，它具有所有八种切割活性。揭示了所有四种类型的向导的利用以及对单链 DNA（ssDNA）和 RNA 靶标的有效切割。进一步发现，5'P 向导的 5' 端核苷酸偏好，但 5'OH 向导没有明显的偏好。此外，向导或向导/靶双链体与 MhAgo 之间的亲和力是影响切割效率的因素之一，这大大损害了中心和 3' 补充区的错配的切割效率。结构和突变分析表明，MhAgo 切割活性背后存在一些未知的独特结构特征。同时，揭示了 5'OH 向导 RNA（gRNA）介导的质粒切割活性。总之，MhAgo 是多功能的，其生化特性提高了我们对 pAgos 和基于 pAgo 的技术的理解。
