Nature doi:10.1038/nature12971

In eukaryotes, Argonaute (Ago) proteins are the 'slicers' of RNA interference (RNAi) pathways and are guided to their RNA targets by complementary small RNAs anchored in the Ago pocket. Ago proteins are also found in prokaryotes, but their functional roles remain puzzling, as archaea and bacteria lack RNAi machinery. A study in 2013 provided evidence that Ago from the bacterium Rhodobacter sphaeroides uses small RNAs and DNAs to target invading plasmid DNA. Swarts et al. now demonstrate that Thermus thermophilus Ago (TtAgo) mediates DNA-guided cleavage of plasmid DNA. The authors found that T. thermophilus strains lacking functional TtAgo exhibit higher transformation efficiencies and plasmid levels than wild-type strains, suggesting that TtAgo regulates uptake or processing of foreign plasmid DNA. TtAgo co-purifies with 5′-phosphorylated DNAs (∼13–25 nucleotides in size), which the authors refer to as 'small interfering DNA' (siDNA). Further analyses revealed that TtAgo–siDNA complexes can cleave complementary single-stranded DNA under in vitro conditions, and TtAgo, complexed with siDNAs derived from invading plasmid sequences, can selectively nick negatively supercoiled plasmids or linearize them if a pair of nicking complexes with nearby targets is used. Though the process by which siDNAs are generated from plasmid DNA remains unknown, the authors propose that DNA-guided DNA interference defines a new bacterial defense pathway that further illustrates the diverse roles of Ago proteins.