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

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.

doi:10.64898/2026.05.04.722794

bioRxiv · 10.64898/2026.05.04.722794

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

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.

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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.

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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.. 2026-05-05. Coordinated synthesis of double-stranded DNA by a dual reverse transcriptase immune system. https://doi.org/10.64898/2026.05.04.722794

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