Coordinated synthesis of double-stranded DNA by a dual reverse transcriptase immune system

Abstract Defense-associated reverse transcriptase (DRT) systems mediate antiviral immunity through distinct modes of cDNA synthesis: Class 1 DRTs catalyze untemplated synthesis, whereas Class 2 DRTs polymerize noncoding RNA-templated products. However, how these distinct modes drive defense remains unclear. Here, we report that DRT3 immunity arises when Class 1 and Class 2 RT activities cooperate to produce self-complementary double-stranded DNA (dsDNA). DRT3a uses a 5′-ACACAC-3′ RNA template to synthesize poly(dTdG) repeats, whereas DRT3b synthesizes poly(dCdA) repeats without any nucleic acid template. Cryo-electron microscopy reveals that DRT3b forms a hexamer and uses active-site-adjacent residues as deoxyadenosine and deoxycytidine gates to enforce alternating nucleotide addition, representing a unique example of amino acid-templated DNA polymerization. DRT3 is toxic in cells lacking RecBCD, implicating host recombination machinery in limiting dsDNA accumulation, and the phage-encoded RecBCD inhibitor Gam triggers DRT3-mediated abortive infection. These findings reveal how two polymerases with distinct templating strategies generate complementary DNA for defense. Competing Interest Statement Columbia University has filed a patent application related to this work. S.H.S. is a co-founder and scientific advisor to Dahlia Biosciences, a scientific advisor to CrisprBits and Prime Medicine, and an equity holder in Dahlia Biosciences and CrisprBits. The remaining authors declare no competing interests. Footnotes This submission reflects the final accepted manuscript, including a revised title and abstract, supplemental tables, supplemental videos, and the Key Resources Table. We have also added new supplemental figures and text exploring DRT3b amino acid templating.