Protein-templated synthesis of dinucleotide repeat DNA by an antiphage reverse transcriptase

Editor’s summary

Bacterial defense systems often deploy unconventional biochemistry to thwart viral infection. Investigating the antiphage system DRT3, Deng et al. found that it defends against infection by synthesizing repetitive poly(GT/AC) double-stranded DNA using two distinct reverse transcriptases (RTs). One of its RTs copies a noncoding RNA, and the second synthesizes the complementary DNA strand de novo without any nucleic acid template. Instead, it uses its own amino acids as a physical mold to enforce precise base alternation. This work reveals a surprising protein-templated mechanism for sequence-specific DNA synthesis, expanding the known repertoire of enzymatic polymerization. —Di Jiang

Abstract

Defense-associated reverse transcriptases (DRTs) are widespread bacterial antiphage systems that use unconventional mechanisms of polynucleotide synthesis. We show that DRT3, which comprises two distinct RTs (Drt3a and Drt3b) and a noncoding RNA (ncRNA), synthesizes alternating poly(GT/AC) double-stranded DNA. Cryo–electron microscopy structures at 2.6-angstrom resolution reveal a D3-symmetric 6:6:6 complex of Drt3a, Drt3b, and ncRNA. Drt3a produces the poly(GT) strand using a conserved ACACAC template within the ncRNA. Notably, Drt3b synthesizes a complementary, protein-primed poly(AC) strand in the complete absence of a nucleic acid template, using conserved active site residues specific to Drt3b to enforce precise base alternation. These findings expand the functional landscape of nucleic acid polymerases, revealing a protein-templated mechanism for sequence-specific DNA synthesis.

Access the full article

View all access options to continue reading this article.

Supplementary Materials

The PDF file includes:

Materials and Methods

Figs. S1 to S14

Table S1

References (47–74)

Download
18.16 MB

Other Supplementary Material for this manuscript includes the following:

MDAR Reproducibility Checklist

Download
121.49 KB

Download
2.08 MB

References and Notes

I. R. Lehman, M. J. Bessman, E. S. Simms, A. Kornberg, Enzymatic synthesis of deoxyribonucleic acid. I. Preparation of substrates and partial purification of an enzyme from Escherichia coli. J. Biol. Chem. 233, 163–170 (1958).

S. B. Weiss, Enzymatic incorporation of ribonucleoside triphosphates into the interpolynucleotide linkages of ribonucleic acid. Proc. Natl. Acad. Sci. U.S.A. 46, 1020–1030 (1960).

D. Baltimore, R. M. Franklin, A new ribonucleic acid polymerase appearing after Mengovirus infection of L-cells. J. Biol. Chem. 238, 3395–3400 (1963).

D. Baltimore, RNA-dependent DNA polymerase in virions of RNA tumour viruses. Nature 226, 1209–1211 (1970).

H. M. Temin, S. Mizutani, RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature 226, 1211–1213 (1970).

M. Edmonds, R. Abrams, Polynucleotide biosynthesis: Formation of a sequence of adenylate units from adenosine triphosphate by an enzyme from thymus nuclei. J. Biol. Chem. 235, 1142–1149 (1960).

R. Aphasizhev, S. Sbicego, M. Peris, S.-H. Jang, I. Aphasizheva, A. M. Simpson, A. Rivlin, L. Simpson, Trypanosome mitochondrial 3′ terminal uridylyl transferase (TUTase): The key enzyme in U-insertion/deletion RNA editing. Cell 108, 637–648 (2002).

F. J. Bollum, Calf thymus polymerase. J. Biol. Chem. 235, 2399–2403 (1960).

F. W. Alt, D. Baltimore, Joining of immunoglobulin heavy chain gene segments: Implications from a chromosome with evidence of three D-JH fusions. Proc. Natl. Acad. Sci. U.S.A. 79, 4118–4122 (1982).

D. T. Nair, R. E. Johnson, L. Prakash, S. Prakash, A. K. Aggarwal, Rev1 employs a novel mechanism of DNA synthesis using a protein template. Science 309, 2219–2222 (2005).

J. Preiss, M. Dieckmann, P. Berg, The enzymic synthesis of amino acyl derivatives of ribonucleic acid. IV. The formation of the 3′-hydroxyl terminal trinucleotide sequence of amino acid-acceptor ribonucleic acid. J. Biol. Chem. 236, 1748–1757 (1961).

J. J. Furth, J. Hurwitz, R. Krug, M. Alexander, The incorporation of adenylic and cytidylic acids into ribonucleic acid. J. Biol. Chem. 236, 3317–3322 (1961).

Y. Xiong, T. A. Steitz, Mechanism of transfer RNA maturation by CCA-adding enzyme without using an oligonucleotide template. Nature 430, 640–645 (2004).

L. Sun, J. Wu, F. Du, X. Chen, Z. J. Chen, Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339, 786–791 (2013).

A. T. Whiteley, J. B. Eaglesham, C. C. de Oliveira Mann, B. R. Morehouse, B. Lowey, E. A. Nieminen, O. Danilchanka, D. S. King, A. S. Y. Lee, J. J. Mekalanos, P. J. Kranzusch, Bacterial cGAS-like enzymes synthesize diverse nucleotide signals. Nature 567, 194–199 (2019).

M. A. Preston, D. F. Porter, F. Chen, N. Buter, C. P. Lapointe, S. Keles, J. Kimble, M. Wickens, Unbiased screen of RNA tailing activities reveals a poly(UG) polymerase. Nat. Methods 16, 437–445 (2019).

A. Shukla, J. Yan, D. J. Pagano, A. E. Dodson, Y. Fei, J. Gorham, J. G. Seidman, M. Wickens, S. Kennedy, poly(UG)-tailed RNAs in genome protection and epigenetic inheritance. Nature 582, 283–288 (2020).

K. K. Kojima, M. Kanehisa, Systematic survey for novel types of prokaryotic retroelements based on gene neighborhood and protein architecture. Mol. Biol. Evol. 25, 1395–1404 (2008).

D. M. Simon, S. Zimmerly, A diversity of uncharacterized reverse transcriptases in bacteria. Nucleic Acids Res. 36, 7219–7229 (2008).

M. R. Mestre, L. A. Gao, S. A. Shah, A. López-Beltrán, A. González-Delgado, F. Martínez-Abarca, J. Iranzo, M. Redrejo-Rodríguez, F. Zhang, N. Toro, UG/Abi: A highly diverse family of prokaryotic reverse transcriptases associated with defense functions. Nucleic Acids Res. 50, 6084–6101 (2022).

E. Emond, B. J. Holler, I. Boucher, P. A. Vandenbergh, E. R. Vedamuthu, J. K. Kondo, S. Moineau, Phenotypic and genetic characterization of the bacteriophage abortive infection mechanism AbiK from Lactococcus lactis. Appl. Environ. Microbiol. 63, 1274–1283 (1997).

C. Wang, M. Villion, C. Semper, C. Coros, S. Moineau, S. Zimmerly, A reverse transcriptase-related protein mediates phage resistance and polymerizes untemplated DNA in vitro. Nucleic Acids Res. 39, 7620–7629 (2011).

M. Figiel, M. Gapińska, M. Czarnocki-Cieciura, W. Zajko, M. Sroka, K. Skowronek, M. Nowotny, Mechanism of protein-primed template-independent DNA synthesis by Abi polymerases. Nucleic Acids Res. 50, 10026–10040 (2022).

X.-Y. Song, Y. Xia, J.-T. Zhang, Y.-J. Liu, H. Qi, X.-Y. Wei, H. Hu, Y. Xia, X. Liu, Y.-F. Ma, N. Jia, Bacterial reverse transcriptase synthesizes long poly(A)-rich cDNA for antiphage defense. Science 388, eads4639 (2025).

S. Tang, R. Žedaveinytė, N. Burman, S. Pandey, J. L. Ramirez, L. M. Kulber, T. Wiegand, R. A. Wilkinson, Y. Ma, D. J. Zhang, G. D. Lampe, M. Berisa, M. Jovanovic, B. Wiedenheft, S. H. Sternberg, Protein-primed homopolymer synthesis by an antiviral reverse transcriptase. Nature 643, 1352–1362 (2025).

J. Han, B. Liu, J. Tang, S. Zhang, X. Wang, X. Li, Q. Zhang, Z. Liu, W. Wang, Y. Liu, R. Zhou, H. Yin, Y. Wei, Z. Li, M. Zhang, Z. Deng, H. Zhang, Non-coding RNA mediates the defense-associated reverse transcriptase (DRT) anti-phage oligomerization transition. EMBO J. 44, 5429–5442 (2025).

M. E. Wilkinson, D. Li, A. Gao, R. K. Macrae, F. Zhang, Phage-triggered reverse transcription assembles a toxic repetitive gene from a noncoding RNA. Science 386, eadq3977 (2024).

S. Tang, V. Conte, D. J. Zhang, R. Žedaveinytė, G. D. Lampe, T. Wiegand, L. C. Tang, M. Wang, M. W. G. Walker, J. T. George, L. E. Berchowitz, M. Jovanovic, S. H. Sternberg, De novo gene synthesis by an antiviral reverse transcriptase. Science 386, eadq0876 (2024).

L. Gao, H. Altae-Tran, F. Böhning, K. S. Makarova, M. Segel, J. L. Schmid-Burgk, J. Koob, Y. I. Wolf, E. V. Koonin, F. Zhang, Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077–1084 (2020).

J. L. Stamos, A. M. Lentzsch, A. M. Lambowitz, Structure of a Thermostable Group II Intron Reverse Transcriptase with Template-Primer and Its Functional and Evolutionary Implications. Mol. Cell 68, 926–939.e4 (2017).

Y. He, H. Song, H. Chan, B. Liu, Y. Wang, L. Sušac, Z. H. Zhou, J. Feigon, Structure of Tetrahymena telomerase-bound CST with polymerase α-primase. Nature 608, 813–818 (2022).

Y. Xiong, T. H. Eickbush, Origin and evolution of retroelements based upon their reverse transcriptase sequences. EMBO J. 9, 3353–3362 (1990).

Y. He, Y. Wang, B. Liu, C. Helmling, L. Sušac, R. Cheng, Z. H. Zhou, J. Feigon, Structures of telomerase at several steps of telomere repeat synthesis. Nature 593, 454–459 (2021).

M. Gapińska, W. Zajko, K. Skowronek, M. Figiel, P. S. Krawczyk, A. A. Egorov, A. Dziembowski, M. J. O. Johansson, M. Nowotny, Structure-functional characterization of Lactococcus AbiA phage defense system. Nucleic Acids Res. 52, 4723–4738 (2024).

Y. Wang, H. Wu, J. Li, Q. An, Z. Tian, Z. Deng, Anti-phage defense mechanism involving phage-encoded DNA binding protein and bacterial reverse transcriptase DRT4. Nat. Commun. 17, 289 (2025).

Y. Wang, H. Wu, J. Li, Z. Tian, Z. Deng, Cryo-EM structure of the bacterial anti-phage defense system DRT6. Biochem. Biophys. Res. Commun. 791, 152955 (2025).

M. Figiel, V. K. Viswanath, M. Czarnocki-Cieciura, M. Šoltysová, J. Rybakowska, A. A. Egorov, V. Hauryliuk, M. J. O. Johansson, M. Nowotny, Structures and enzymatic mechanisms of DRT7/UG10 antiphage reverse transcriptases. bioRxiv 2026.02.16.706125 [Preprint] (2026); https://doi.org/10.64898/2026.02.16.706125.

S. Kim, H. Xu, G. Mohr, J. Yao, Y. J. Zhang, G. Cui, A. M. Lambowitz, A bacterial PrimPol-reverse transcriptase hybrid protein has a proofreading exonuclease activity that can be transferred to other reverse transcriptases. bioRxiv 2025.09.07.674619 [Preprint] (2025); https://doi.org/10.1101/2025.09.07.674619.

S. Tang, J. L. Ramirez, M. R. Mestre, D. J. Zhang, M. Wang, T. Wiegand, Y. Ma, M. Jovanovic, R. Pinilla-Redondo, S. H. Sternberg, Antiviral reverse transcriptases reveal the evolutionary origin of telomerase. bioRxiv 2025.10.16.682844 [Preprint] (2025); https://doi.org/10.1101/2025.10.16.682844.

H. K. Schachman, J. Adler, C. M. Radding, I. R. Lehman, A. Kornberg, Enzymatic synthesis of deoxyribonucleic acid. VII. Synthesis of a polymer of deoxyadenylate and deoxythymidylate. J. Biol. Chem. 235, 3242–3249 (1960).

N. Ogata, T. Miura, Genetic information ‘created’ by archaebacterial DNA polymerase. Biochem. J. 324, 667–671 (1997).

K. Hanaki, T. Odawara, T. Muramatsu, Y. Kuchino, M. Masuda, K. Yamamoto, C. Nozaki, K. Mizuno, H. Yoshikura, Primer/template-independent synthesis of poly d(A-T) by Taq polymerase. Biochem. Biophys. Res. Commun. 238, 113–118 (1997).

N. V. Zyrina, V. N. Antipova, L. A. Zheleznaya, Ab initiosynthesis by DNA polymerases. FEMS Microbiol. Lett. 351, 1–6 (2014).

G. Levinson, G. A. Gutman, Slipped-strand mispairing: A major mechanism for DNA sequence evolution. Mol. Biol. Evol. 4, 203–221 (1987).

S. Roschdi, T. Kume, R. J. Petersen, A. McCann, C. A. Escobar, A. Richard, S. E. Butcher, Sequence and ionic requirements of pUG fold quadruplexes. bioRxiv 2025.10.28.685102 [Preprint] (2025); https://doi.org/10.1101/2025.10.28.685102.

A. M. Burroughs, D. Zhang, D. E. Schäffer, L. M. Iyer, L. Aravind, Comparative genomic analyses reveal a vast, novel network of nucleotide-centric systems in biological conflicts, immunity and signaling. Nucleic Acids Res. 43, 10633–10654 (2015).

S. F. Altschul, T. L. Madden, A. A. Schäffer, J. Zhang, Z. Zhang, W. Miller, D. J. Lipman, Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).

M. Steinegger, J. Söding, MMseqs2 enables sensitive protein sequence searching for the analysis of massive data sets. Nat. Biotechnol. 35, 1026–1028 (2017).

K. Katoh, D. M. Standley, MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).

S. Capella-Gutiérrez, J. M. Silla-Martínez, T. Gabaldón, trimAl: A tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25, 1972–1973 (2009).

L.-T. Nguyen, H. A. Schmidt, A. von Haeseler, B. Q. Minh, IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274 (2015).

I. Letunic, P. Bork, Interactive Tree of Life (iTOL) v6: Recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Res. 52 (W1), W78–W82 (2024).

L. J. Revell, phytools 2.0: An updated R ecosystem for phylogenetic comparative methods (and other things). PeerJ 12, e16505 (2024).

G. E. Crooks, G. Hon, J.-M. Chandonia, S. E. Brenner, WebLogo: A sequence logo generator. Genome Res. 14, 1188–1190 (2004).

S. Picelli, A. K. Björklund, B. Reinius, S. Sagasser, G. Winberg, R. Sandberg, Tn5 transposase and tagmentation procedures for massively scaled sequencing projects. Genome Res. 24, 2033–2040 (2014).

M. Martin, Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. J. 17, 10 (2011).

M. Vasimuddin, S. Misra, H. Li, S. Aluru, “ Efficient architecture-aware acceleration of BWA-MEM for multicore systems,” 2019 IEEE International Parallel and Distributed Processing Symposium (IPDPS), Rio de Janeiro, Brazil, 20 to 24 May 2019 (IEEE, 2019); https://ieeexplore.ieee.org/document/8820962/.

P. Danecek, J. K. Bonfield, J. Liddle, J. Marshall, V. Ohan, M. O. Pollard, A. Whitwham, T. Keane, S. A. McCarthy, R. M. Davies, H. Li, Twelve years of SAMtools and BCFtools. Gigascience 10, giab008 (2021).

T. L. Bailey, M. Boden, F. A. Buske, M. Frith, C. E. Grant, L. Clementi, J. Ren, W. W. Li, W. S. Noble, MEME SUITE: Tools for motif discovery and searching. Nucleic Acids Res. 37, W202–W208 (2009).

T. J. Wheeler, S. R. Eddy, nhmmer: DNA homology search with profile HMMs. Bioinformatics 29, 2487–2489 (2013).

E. P. Nawrocki, S. R. Eddy, Infernal 1.1: 100-fold faster RNA homology searches. Bioinformatics 29, 2933–2935 (2013).

E. Rivas, J. Clements, S. R. Eddy, A statistical test for conserved RNA structure shows lack of evidence for structure in lncRNAs. Nat. Methods 14, 45–48 (2017).

C. E. Grant, T. L. Bailey, W. S. Noble, FIMO: Scanning for occurrences of a given motif. Bioinformatics 27, 1017–1018 (2011).

A. Punjani, J. L. Rubinstein, D. J. Fleet, M. A. Brubaker, cryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

T. Bepler, A. Morin, M. Rapp, J. Brasch, L. Shapiro, A. J. Noble, B. Berger, Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs. Nat. Methods 16, 1153–1160 (2019).

R. W. Grosse-Kunstleve, N. K. Sauter, N. W. Moriarty, P. D. Adams, TheComputational Crystallography Toolbox: Crystallographic algorithms in a reusable software framework. J. Appl. Cryst. 35, 126–136 (2002).

K. Jamali, L. Käll, R. Zhang, A. Brown, D. Kimanius, S. H. W. Scheres, Automated model building and protein identification in cryo-EM maps. Nature 628, 450–457 (2024).

P. Emsley, K. Cowtan, Coot: Model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

P. D. Adams, P. V. Afonine, G. Bunkóczi, V. B. Chen, I. W. Davis, N. Echols, J. J. Headd, L.-W. Hung, G. J. Kapral, R. W. Grosse-Kunstleve, A. J. McCoy, N. W. Moriarty, R. Oeffner, R. J. Read, D. C. Richardson, J. S. Richardson, T. C. Terwilliger, P. H. Zwart, PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

S. Srikant, C. K. Guegler, M. T. Laub, The evolution of a counter-defense mechanism in a virus constrains its host range. eLife 11, e79549 (2022).

D. E. Deatherage, J. E. Barrick, Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq. Methods Mol. Biol. 1151, 165–188 (2014).

R. Odegrip, A. S. Nilsson, E. Haggård-Ljungquist, Identification of a gene encoding a functional reverse transcriptase within a highly variable locus in the P2-like coliphages. J. Bacteriol. 188, 1643–1647 (2006).

C. Hill, L. A. Miller, T. R. Klaenhammer, Nucleotide sequence and distribution of the pTR2030 resistance determinant (hsp) which aborts bacteriophage infection in lactococci. Appl. Environ. Microbiol. 56, 2255–2258 (1990).

P. K. Dinsmore, T. R. Klaenhammer, Phenotypic Consequences of Altering the Copy Number of abiA, a Gene Responsible for Aborting Bacteriophage Infections in Lactococcus lactis. Appl. Environ. Microbiol. 60, 1129–1136 (1994).