Modified DNA substrate selectivity by GmrSD-family Type IV restriction enzyme BrxU

Bacteriophages (phages), viral predators of bacteria, generate selection pressure that causes bacteria to evolve defence systems. Type I, II and III restriction enzymes cleave incoming non-modified phage DNAs. Phages have evolved to defend against these restriction systems by modifying their DNA so that they are no longer suitable substrates. Type IV restriction enzymes have evolved to recognize and cleave modified DNA. We have recently characterized and solved the first structure for the Type IV GmrSD-family enzymes, using the BrxU homologue from Escherichia fergusonii . Though promiscuous in target modifications, little is known about BrxU substrate preference. We used modified DNAs in in vitro assays to characterize the substrate preferences of BrxU and investigate the impact of the GmrSD-inhibitor IPI* on BrxU activity. These data extend our knowledge of phage–host interactions and inform mechanistic studies on the reaction cycle of BrxU and GmrSD homologues. This article is part of the discussion meeting issue ‘The ecology and evolution of bacterial immune systems’.