GEOapplication/xmlftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE329nnn/GSE329894/primaryOK200OtherEscherichia coliOtherhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE329894GEOGSEfalseCoordinated synthesis of double-stranded DNA by a dual reverse transcriptase immune system [Tn-seq]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 a distinctive 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. The cryo-electron microscopy structure reveals 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.2026/06/22GSE329894GSM9712830GSM9712831GSM9712820GSM9712832GSM9712821GSM9712833GSM9712822GSM9712834GSM9712823GSM9712835GSM9712824GSM9712825GSM9712836GSM9712837GSM9712826GSM9712838GSM9712827GSM9712828GSM9712829GSM971281935738329894Escherichia coli