Antiviral reverse transcriptase–primase synthesizes protein-templated DNA

Abstract Defense-associated reverse transcriptases (DRTs) are widespread in bacteria1,2, but how multi-domain DRTs containing RT and additional catalytic activities coordinate antiviral defense remains unclear. Here we show that DRT7, which contains both reverse transcriptase (RT) and primase–polymerase (PP) domains, provides broad-spectrum anti-phage immunity through abortive infection and can be activated by a phage-encoded putative transcriptional regulator. Upon activation, DRT7 synthesizes long, protein-primed, palindromic poly(A)/poly(T)-rich duplex-like DNA. Cryo–electron microscopy structures reveal that RT initiates protein-primed, protein-templated, sequence-specific poly(T) synthesis through an arginine-rich recognition pocket without requiring a complementary nucleic acid template, thereby converting DRT7 from an inactive closed dimer to an active open dimer. The RT-produced poly(T) then serves as both primer and template for PP-mediated poly(A) extension, with iterative handoff between RT and PP generating palindromic, alternating poly(A)/poly(T) ssDNA tracts that assemble into fold-back duplex-like DNA. These findings uncover an unexpected antiviral strategy based on protein self-templating, sequence-specific duplex-like DNA synthesis and reveal how coupling RTs with additional catalytic activities expands the functional scope of nucleic acid synthesis pathways. Competing Interest Statement The authors have declared no competing interest.