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

Prokaryotic Argonaute proteins (pAgos) are homologs of eukaryotic Argonautes involved in RNA interference that regulate gene expression and play a role in cell defense against invaders. However, little is known about the functional activities and target specificity of various groups of pAgos but these proteins already found many applications in molecular biology. Here, we describe five pAgo proteins from mesophilic bacteria which act as DNA-guided DNA endonucleases, and analyze their ability to target chromosomal and invader DNA. In vitro, studied proteins preferentially use small guide DNAs for precise cleavage of single-stranded DNA at a wide range of temperatures, and can also process double-stranded DNA. During their expression in Escherichia coli, all five pAgos are loaded with small DNA guides with preferences for plasmid DNA and for 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, but not other pAgos, also protects the cells from bacteriophage infection and is preferentially loaded with phage-derived guide DNAs, thus preventing phage replication. This suggests that the ability of catalytically active pAgos to target multicopy elements is important for their protective function. New pAgos wider our knowledge about diversity of these proteins and may become new instruments for applied research.