Members of the conservedArgonaute protein family use small RNA guides to find their mRNA targets to regulate gene expression and suppress mobile genetic elements in eukaryotes. Argonautes are also present in many bacterial and archaeal species 1-3 . Unlike eukaryotic proteins, several studied prokaryotic Argonautes use small DNA guides to cleave DNA, a process dubbed DNA interference 4-8 . 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 CbAgo and demonstrated that it induces cleavage of multicopy genetic elements, including plasmids, transposons and repetitive chromosomal loci. Generation of small DNA guides employed by CbAgo requires cooperation between its intrinsic endonuclease activity and the cellular double-strand break repair machinery. The mechanism of guide generation ensures that small DNA guides are enriched in sequences that target foreign DNA and endows CbAgo with capacity to eliminate plasmids and fight phage infection. Similar principles may underlie the specificity of self-nonself discrimination by diverse defense systems in prokaryotes.Argonaute (Ago) proteins are a ubiquitous family of guide-dependent nucleases found in all three domains of life 1,3,9,10 . Eukaryotic Ago proteins (eAgos) are RNA-dependent RNA nucleases that suppress gene expression by binding to their RNA targets in the cytoplasm or in the nucleus 11,12 . Prokaryotic Agos (pAgos) are extremely diverse in comparison with eAgos and are randomly distributed among bacterial and archaeal lineages suggesting their active spread by horizontal gene transfer 1-3 . In contrast to their eukaryotic counterparts, several studied pAgos were shown to act in vitro as nucleases with distinct specificity toward DNA targets [4][5][6]8,[13][14][15][16][17] . The majority of studied pAgos also prefer to use short single-stranded DNA molecules as guides for their endonuclease activity, however, the molecular mechanism of DNA guide generation in bacterial cells has remained poorly understood. Two pAgo proteins studied in vivo were shown to decrease plasmid content and transformation efficiency in bacterial cells 7,18 , leading to the hypothesis that pAgos use DNA interference to protect the cells against foreign DNA. However, the ability of pAgos to fight genuine
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