Semirandom DNA adducts regulate a filamentous defence-associated reverse transcriptase

Retrons and several defence-associated reverse transcriptases (DRTs) synthesize non-genomic DNA 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 antiphage defence system. We show that DRT1 performs template-free, protein-primed DNA synthesis to generate semirandom DNA adducts. DNA synthesis activates the nitrilase domain of DRT1 while DNA adducts drive assembly of quiescent DRT1 filaments. Filamentous DRT1 is comprised of domain-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 DRT1 activity. Functionally, DRT1 resembles a minimal retron where a single gene produces an RT, effector, and non-genomic antitoxin DNA.