DarHTG is a tripartite phage defence system for purine DNA base ADP-ribosylation

Bacteria evolved a variety of phage defence strategies to combat the constant onslaught of viral threats. DarTG toxin-antitoxin systems provide phage defence by ADP-ribosylation of bases in single-stranded DNA which interferes with viral replication and aborts phage infection. Here, we describe DarHTG, a system widespread among bacteria, that combines DarTG with a non-processive superfamily I helicase functioning as co-toxin for DarT and thereby enabling the ADP-ribosylation of double-stranded DNA. We identified two such DarHTG subclasses which catalyse the ADP-ribosylation of adenosine and guanosine bases, respectively, and reverse these modifications with structurally unique ARH superfamily antitoxins. Thus, DarHTG antitoxins expand the known macromolecular target spectrum of the ARH enzyme class to nucleic acids. These DarHTG systems confer resistance to a broad range of bacteriophages and can be triggered by the highly charged tails of viral single-stranded DNA-binding proteins, which occlude the active site of the ARH antitoxin.