Antiviral reverse transcriptase-primase synthesizes protein-templated DNA

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.