Single-site DNA methylation activates retron defence by enabling effector tetramerisation
- 1European Molecular Biology Laboratory (EMBL)
Abstract
Retrons are unique among bacterial defence systems in using DNA to sense phage infection. They achieve this by producing multicopy single-stranded DNA (msDNA) that monitors the activity of DNA-targeting phage proteins. Although we understand how extensive msDNA perturbations such as degradation activate retrons, activation by subtle chemical modifications remains enigmatic. Here, we used a structure-function approach to elucidate how single-site msDNA methylation activates Retron-Sen2. The cryo-EM structure of Retron-Sen2 features RcaT, the most prevalent retron effector, sequestered as an inactive dimer within a four-component assembly of previously undescribed architecture. Methylation by DNA adenine methyltransferase disrupts the RcaT-msDNA interaction and exposes a previously occluded interface that enables RcaT dimers to form tetramers, leading to retron disassembly. We show that RcaT dimer-dimer assembly is essential for its toxicity. To assess whether this is a conserved RcaT feature, we characterised additional homologues. RcaTs capable of tetramerisation or higher-order oligomerisation are toxic and form distinct phylogenetic clades from RcaTs that remain dimeric and are non-toxic. Together, our findings provide a paradigm for retron activation by single-site msDNA modifications and showcase how variation in an intrinsic protein property can diversify the toxicity of the same bacterial defence effector during evolution.
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BibTeX
@misc{spatharas2026singlesite,
title = {{Single-site DNA methylation activates retron defence by enabling effector tetramerisation}},
author = {P. Spatharas and A. L. J. Yang and E. Mullapudi and G. Chojnowski and F. Corona and D. Kumar and J. Bobonis and A. Typas and M. Wilmanns},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.13.758329},
url = {https://www.biorxiv.org/content/10.64898/2026.10.13.758329v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Single-site DNA methylation activates retron defence by enabling effector tetramerisation
AU - P. Spatharas
AU - A. L. J. Yang
AU - E. Mullapudi
AU - G. Chojnowski
AU - F. Corona
AU - D. Kumar
AU - J. Bobonis
AU - A. Typas
AU - M. Wilmanns
PY - 2026
DA - 2026/10/13
PB - bioRxiv
DO - 10.64898/2026.10.13.758329
UR - https://www.biorxiv.org/content/10.64898/2026.10.13.758329v1
AB - Retrons are unique among bacterial defence systems in using DNA to sense phage infection. They achieve this by producing multicopy single-stranded DNA (msDNA) that monitors the activity of DNA-targeting phage proteins. Although we understand how extensive msDNA perturbations such as degradation activate retrons, activation by subtle chemical modifications remains enigmatic. Here, we used a structure-function approach to elucidate how single-site msDNA methylation activates Retron-Sen2. The cryo-EM structure of Retron-Sen2 features RcaT, the most prevalent retron effector, sequestered as an inactive dimer within a four-component assembly of previously undescribed architecture. Methylation by DNA adenine methyltransferase disrupts the RcaT-msDNA interaction and exposes a previously occluded interface that enables RcaT dimers to form tetramers, leading to retron disassembly. We show that RcaT dimer-dimer assembly is essential for its toxicity. To assess whether this is a conserved RcaT feature, we characterised additional homologues. RcaTs capable of tetramerisation or higher-order oligomerisation are toxic and form distinct phylogenetic clades from RcaTs that remain dimeric and are non-toxic. Together, our findings provide a paradigm for retron activation by single-site msDNA modifications and showcase how variation in an intrinsic protein property can diversify the toxicity of the same bacterial defence effector during evolution.
ER -