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

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

Pujuan Deng https://orcid.org/0000-0002-3197-3126,

Hyunbin Lee https://orcid.org/0009-0006-1911-435X,

Carlo Armijo https://orcid.org/0009-0007-9234-7413,

Haoqing Wang https://orcid.org/0000-0003-0277-3018, and

Alex Gao https://orcid.org/0000-0002-3579-0327

Science

16 Apr 2026

First Release

DOI: 10.1126/science.aed1656

Alex Gao Lab:

https://med.stanford.edu/profiles/alex-gao#bio

1. Background

DNA and RNA polymerases are traditionally classified into two groups: template-dependent enzymes, which copy existing nucleic acid sequences, and template-independent enzymes, which typically produce low-complexity or random sequences. Recently, bacterial defense-associated reverse transcriptases (DRTs) have emerged as a diverse class of enzymes involved in anti-phage immunity, exhibiting unusual polymerization activities. However, whether polymerases can generate sequence-defined DNA without a nucleic acid template has remained largely unknown.

2. Objective

This study aims to characterize the molecular mechanism of the DRT3 system, focusing on how it synthesizes DNA and contributes to bacterial anti-phage defense.

3. Methods

• Molecular biology & biochemistry: cloning, heterologous expression in E. coli, in vitro polymerization assays
• Sequencing approaches: tagmentation sequencing to identify DNA products
• Cryo-electron microscopy (cryo-EM): high-resolution structural determination (2.6 Å)
• Mutagenesis: functional dissection of catalytic residues
• Phage assays: evaluation of anti-phage activity

4. Key Results

4.1 Unique DNA product

The DRT3 system synthesizes double-stranded DNA composed of alternating dinucleotide repeats: poly(GT/AC), produced independently of phage infection.

4.2 Division of enzymatic functions

DRT3 consists of two reverse transcriptases with distinct mechanisms:

• Drt3a
  • Uses a noncoding RNA (ncRNA) template
  • Synthesizes poly(GT) single-stranded DNA
  • Mechanistically resembles telomerase-like RNA-templated polymerization
• Drt3b
  • Functions without any nucleic acid template
  • Produces poly(AC) DNA
  • Uses specific amino acid residues as a “protein template” to enforce strict base alternation
  • Initiates synthesis via protein-priming (tyrosine-mediated covalent linkage)

The two strands anneal to form a complementary dsDNA duplex.

4.3 Structural organization

Cryo-EM structures reveal a D3-symmetric 6:6:6 ribonucleoprotein complex composed of Drt3a, Drt3b, and ncRNA. The structure shows coordinated synthesis of both DNA strands and positioning of the DNA duplex around the complex.

4.4 Mechanistic novelty

• Drt3a follows canonical template-directed synthesis
• Drt3b represents a previously unknown mechanism:
  • No template strand
  • Sequence specificity encoded by protein–DNA interactions
  • Precise alternation (A–C) enforced by conserved residues (e.g., Glu26, Arg253)

4.5 Biological function

• DRT3 confers robust resistance to bacteriophage infection
• Activation requires a phage protein (ST61)
• Both DNA strands are essential for defense, indicating that the repetitive dsDNA product is functionally important

5. Significance and Implications

Conceptual advances

• Demonstrates sequence-specific DNA synthesis without a nucleic acid template
• Introduces a new paradigm:
  protein-templated nucleic acid synthesis
• Expands the functional diversity of polymerases and reverse transcriptases

Biological implications

• Reveals a novel anti-phage defense strategy
• Suggests DNA products may form higher-order structures or act as molecular decoys

Technological potential

• Opens avenues for:
  • Synthetic biology (programmable DNA synthesis systems)
  • Protein-engineered polymerases
  • Biomolecular nanomaterials with repetitive DNA architectures

6. Conclusion

The DRT3 system represents a fundamentally new class of nucleic acid polymerization machinery, in which two distinct enzymes cooperate to generate a defined DNA duplex using both RNA-templated and protein-templated mechanisms. This discovery challenges conventional views of genetic information transfer and highlights the evolutionary innovation of bacterial defense systems.

Original Paper: 

https://www.science.org/doi/10.1126/science.aed1656

Alex Gao Lab: 

https://med.stanford.edu/profiles/alex-gao#bio