Phage-encoded factor stimulates DNA degradation by the Hna anti-phage defense system

Prokaryotic organisms have evolved unique strategies to acquire immunity against the constant threat of bacteriophage (phage) and mobile genetic elements. Hna is a broadly distributed anti-phage immune system that confers resistance against diverse phage by eliciting an abortive infection response. Using a combination of biochemistry, cryo-electron microscopy, and single-molecule fluorescence imaging, we reveal that Hna functions as a 3'-5' single-stranded DNA exonuclease that forms an auto-inhibited dimer under physiological ATP concentrations. Biochemical and mutational analyses demonstrate that Hna catalytic outputs are governed by kinetic partitioning between ATPase and nuclease active sites. Disruption of this balance enhances DNA cleavage and causes cellular toxicity. Furthermore, we show that a phage-encoded single-stranded DNA-binding protein (5 A SSB) destabilizes the autoinhibited Hna dimer and shifts catalytic partitioning toward dysregulated nuclease activation. Conversely, phage escape mutants encode SSB variants that evade Hna surveillance by adopting higher order stoichiometries with enhanced DNA binding affinity. Our work establishes the molecular basis of Hna-mediated anti-phage activity and provides insights into how phage-encoded proteins can directly stimulate a bacterial immune response.