Semirandom DNA adducts regulate a filamentous defence-associated reverse transcriptase
Retrons and several defence-associated reverse transcriptases (DRTs) synthesize non-genomicDNA for bacteriophage immunity. In some instances, this non-genomic DNA is of undefined,semirandom sequence. How undefined DNA sequences impart antiphage defence is not known.Here we report the cryo-EM structure and functional characterization of the DRT1 antiphagedefence system. We show that DRT1 performs template-free, protein-primed DNA synthesis togenerate semirandom DNA adducts. DNA synthesis activates the nitrilase domain of DRT1 whileDNA adducts drive assembly of quiescent DRT1 filaments. Filamentous DRT1 is comprised ofdomain-swapped C-termini that are entwined, forming pseudoknots between tetrameric stacks.This configuration occludes the apo-nitrilase active site, resulting in a dormant state.Bacteriophage escape mutants identify a T4 single-stranded DNA helicase required for DRT1activity. Functionally, DRT1 resembles a minimal retron where a single gene produces an RT,effector, and non-genomic antitoxin DNA.