DNA targeting and interference by a bacterial Argonaute nuclease

Members of the conserved Argonaute protein family use small RNA guides to locate their mRNA targets and regulate gene expression and suppress mobile genetic elements in eukaryotes 1 , 2 . Argonautes are also present in many bacterial and archaeal species 3 – 5 . Unlike eukaryotic proteins, several prokaryotic Argonaute proteins use small DNA guides to cleave DNA, a process known as DNA interference 6 – 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 analyse the activity of a bacterial Argonaute nuclease from Clostridium butyricum ( Cb Ago) in vivo. We show that Cb Ago targets multicopy genetic elements and suppresses the propagation of plasmids and infection by phages. Cb Ago induces DNA interference between homologous sequences and triggers DNA degradation at double-strand breaks in the target DNA. The loading of Cb Ago with locus-specific small DNA guides depends on both its intrinsic endonuclease activity and the cellular double-strand break repair machinery. A similar interaction was reported for the acquisition of new spacers during CRISPR adaptation, and prokaryotic genomes that encode Ago nucleases are enriched in CRISPR–Cas systems. These results identify molecular mechanisms that generate guides for DNA interference and suggest that the recognition of foreign nucleic acids by prokaryotic defence systems involves common principles.