Wang, M.; Yoneyama, K.; Zedaveinyte, R.; Ishikawa, J.; Tang, S.; Le, H. C.; Wiegand, T.; Ramirez, J. L.; Nagahata, N.; Ma, Y.; Zhang, D. J.; Helmeczi, E.; Berisa, M.; Jovanovic, M.; Hiraizumi, M.; Yamashita, K.; Nishimasu, H.; Sternberg, S. H.

Recent studies have revealed that defense-associated reverse transcriptase (DRT) systems mediate antiviral immunity through distinct modes of cDNA synthesis. Class I DRTs catalyze untemplated DNA synthesis with random or nucleotide-biased sequences, whereas Class II DRTs polymerize noncoding RNA-templated products, including concatemeric repeats and homopolymeric cDNA. However, how these distinct modes of cDNA synthesis are employed to drive antiviral defense remains poorly understood. Here, we report an unprecedented mechanism of DRT3 immunity, in which RT enzymes from both Class I and Class II coordinate their diverse activities to produce self-complementary double-stranded DNA (dsDNA). Remarkably, whereas the DRT3a enzyme relies on a 5'-ACACAC-3' RNA template to synthesize long poly-(dTdG) repeats, DRT3b synthesizes precise poly-(dCdA) repeats without any nucleic acid template at all. Cryo-electron microscopy structures reveal that DRT3b assembles into a hexameric complex and employs active site-adjacent residues to function as deoxyadenosine and deoxycytidine gates that enforce alternating addition to produce dinucleotide repeats, representing a unique example of amino acid-templated DNA polymerization. Strikingly, DRT3 immune systems are toxic in a genetic background lacking E. coli RecBCD, implicating host recombination machinery in limiting DRT3-mediated dsDNA levels. Consistent with this model, we discovered that the phage-encoded RecBCD inhibitor, Gam, potently triggers DRT3-mediated abortive infection. Collectively, our findings reveal how two polymerases with distinct templating strategies cooperate to generate complementary DNA and drive antiviral defense.