Members of the conserved Argonaute protein family use small RNA guides to find their mRNA targets to regulate gene expression and suppress mobile genetic elements in eukaryotes1,2. Argonautes are also present in many bacterial and archaeal species3-5. Unlike eukaryotic proteins, several studied prokaryotic Argonautes use small DNA guides to cleave DNA, a process dubbed DNA interference6-10. However, the natural functions and targets of DNA interference are poorly understood, and the mechanisms of DNA guide generation and target discrimination remain unknown. Here, we studied the in vivo activities of a bacterial Argonaute nuclease from Clostridium butyricum (CbAgo). We demonstrated that CbAgo targets multicopy genetic elements and suppresses propagation of plasmids and infection by phages. CbAgo induces DNA interference between homologous sequences and triggers DNA degradation at double-strand breaks in the target DNA. Loading of CbAgo with locus-specific small DNA guides depends on both its intrinsic endonuclease activity and the cellular double-strand break repair machinery. A similar interplay was reported for acquisition of new spacers during CRISPR adaptation, and prokaryotic genomes encoding pAgo nucleases are enriched in CRISPR-Cas systems. These results identify molecular mechanisms that generate guides for DNA interference and suggest common principles of recognition of foreign nucleic acids by prokaryotic defense systems.

中文翻译：

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细菌 Argonaute 核酸酶的 DNA 靶向和干扰

保守的 Argonaute 蛋白家族的成员使用小 RNA 指南来寻找其 mRNA 靶标，以调节基因表达并抑制真核生物中的移动遗传元件 1,2。Argonautes 也存在于许多细菌和古细菌物种中3-5。与真核蛋白质不同，一些经过研究的原核生物 Argonautes 使用小的 DNA 向导来切割 DNA，这一过程被称为 DNA 干扰 6-10。然而，人们对 DNA 干扰的自然功能和目标知之甚少，DNA 指导生成和目标区分的机制仍然未知。在这里，我们研究了来自丁酸梭菌 (CbAgo) 的细菌 Argonaute 核酸酶的体内活性。我们证明了 CbAgo 靶向多拷贝遗传元件并抑制质粒的传播和噬菌体的感染。CbAgo 诱导同源序列之间的 DNA 干扰，并在目标 DNA 的双链断裂处触发 DNA 降解。用基因座特异性小 DNA 引导加载 CbAgo 取决于其内在核酸内切酶活性和细胞双链断裂修复机制。据报道，在 CRISPR 适应过程中获得新的间隔区也有类似的相互作用，编码 pAgo 核酸酶的原核基因组在 CRISPR-Cas 系统中得到丰富。这些结果确定了生成 DNA 干扰指南的分子机制，并提出了原核防御系统识别外来核酸的共同原则。用基因座特异性小 DNA 引导加载 CbAgo 取决于其内在核酸内切酶活性和细胞双链断裂修复机制。据报道，在 CRISPR 适应过程中获得新的间隔区也有类似的相互作用，编码 pAgo 核酸酶的原核基因组在 CRISPR-Cas 系统中得到丰富。这些结果确定了生成 DNA 干扰指南的分子机制，并提出了原核防御系统识别外来核酸的共同原则。用基因座特异性小 DNA 引导加载 CbAgo 取决于其内在核酸内切酶活性和细胞双链断裂修复机制。据报道，在 CRISPR 适应过程中获得新的间隔区也有类似的相互作用，编码 pAgo 核酸酶的原核基因组在 CRISPR-Cas 系统中得到丰富。这些结果确定了生成 DNA 干扰指南的分子机制，并提出了原核防御系统识别外来核酸的共同原则。