Dual sensing activates antiviral reverse transcriptase for membrane targeting.

The prokaryotic type 2 defense-associated reverse transcriptase (DRT2) system mediates antiviral defense by catalyzing the rolling-circle reverse transcription of a noncoding RNA (ncRNA) and producing the toxic Neo protein that arrests bacterial growth. However, the mechanisms underlying DRT2 activation and effector function remain unknown. Here, we identified two distinct activation mechanisms: direct binding of a phage-encoded single-stranded DNA-binding protein (SSB or SSAP), and the detection of elevated intracellular dGTP levels induced by the phage-encoded ribonucleotide reductase NrdAB. Upon activation, the produced Neo protein directly targets the bacterial membrane, inducing membrane depolarization and growth arrest. Cryo-electron microscopy (cryo-EM) structures of the DRT2-ncRNA complex in its arrested and dGTP-bound active states provide mechanistic insights into rolling-circle ccDNA synthesis and template jumping. Furthermore, these identified activation mechanisms enable the DRT2 system with an engineered ncRNA template to produce a large-scale, user-defined double-stranded DNA (dsDNA) template in vivo, highlighting its potential in biotechnological applications.