Nuclease-NTPase antiphage defence systems use conserved molecular features to control bacterial immunity.

Bacteria encode diverse defence systems, including restriction-modification and CRISPR-Cas, that cleave nucleic acid to protect against phage infection. Bioinformatic analyses demonstrate that many recently identified antiphage defence operons comprise a nuclease and NTPase protein, suggesting that additional nucleic acid-targeting systems remain to be understood. Here we develop large-scale comparative cell biology and biochemical approaches to analyse 16 nuclease-NTPase systems and define molecular features that control antiphage defence. Purification, biochemical characterization and in vitro reconstitution of nucleic acid degradation demonstrates that protein-protein complex formation is a shared feature of multigene nuclease-NTPase systems. We show that PaAbpAB, BtHachiman and EcPD-T4-8 system nucleases use highly degenerate recognition site preferences to enable broad nucleic acid degradation, and the Azaca system exhibits specific phage targeting through the recognition of modified phage genomic DNA. Our results uncover principles of antiphage defence system function and highlight the mechanistic diversity of nuclease-NTPase systems in bacterial immunity.