Abstract Prokaryotic Argonaute proteins (pAgos) are homologs of eukaryotic Argonautes (eAgos) that were similarly proposed to play a role in cell defense against invaders. However, pAgos are much more diverse than eAgos and very little is known about their functional activity and target specificity in vivo . Here, we describe five pAgo proteins from mesophilic bacteria that act as DNA-guided DNA endonucleases and analyze their ability to target chromosomal and invader DNA. In vitro , the analyzed proteins use small guide DNAs for precise cleavage of single-stranded DNA at a wide range of temperatures. Upon their expression in Escherichia coli , all five pAgos are loaded with small DNAs preferentially produced from plasmid DNA and from chromosomal regions of replication termination. One of the tested pAgos, EmaAgo from Exiguobacterium marinum can induce DNA interference between multicopy sequences resulting in targeted processing of homologous plasmid and chromosomal loci. EmaAgo also protects bacteria from bacteriophage infection and is preferentially loaded with phage guide DNAs suggesting that the ability of pAgos to target multicopy elements may be crucial for their protective function. The wide spectrum of pAgo activities suggests that they may have diverse functions in vivo and paves the way for their use in biotechnology.

Bacterial Argonaute nucleases reveal different modes of DNA targeting in vitro and in vivo