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Panos Spatharas @pspatharas.bsky.social · 14/10/2026

11/11 Thanks to all our co-authors: @alessioyang.bsky.social @edumullapudi.bsky.social @gchojnowski.bsky.social Federico Corona, Devansh Kumar and Jacob Bobonis.

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Panos Spatharas @pspatharas.bsky.social · 14/10/2026

10/11 Together, our findings explain how retrons respond to single-site DNA modifications and showcase how the same bacterial defence effector can diversify its toxicity during evolution.

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Panos Spatharas @pspatharas.bsky.social · 14/10/2026

9/11 Across the homologues we examined, RcaTs that form tetramers or larger assemblies are toxic and occupy distinct phylogenetic clades from non-toxic, dimeric RcaTs. This links differences in toxicity to an intrinsic protein property: the capacity to oligomerise.

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Panos Spatharas @pspatharas.bsky.social · 14/10/2026

8/11 This led us to a broader question. Why are some RcaT homologues toxic for the host, while others defend against phages without being toxic on their own? Does their capacity to assemble beyond a dimer explain this difference?

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Panos Spatharas @pspatharas.bsky.social · 14/10/2026

7/11 Together, our findings support a model in which methylation promotes msDNA disengagement, exposing the previously occluded dimer–dimer interface. This allows RcaT tetramerisation and activation, coupled to retron complex disassembly.

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Panos Spatharas @pspatharas.bsky.social · 14/10/2026

6/11 All these assemblies form through the same dimer–dimer interface (a), which was occluded in the intact retron complex. Mutations that disrupt this interface abolish RcaT toxicity (b), showing that assembly beyond a dimer is essential for its activity.

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Panos Spatharas @pspatharas.bsky.social · 14/10/2026

5/11 We next examined RcaT after release. It forms tetramers consisting of two stacked dimers, with additional dimers stacking to form higher-order oligomers.

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Panos Spatharas @pspatharas.bsky.social · 14/10/2026

4/11 In these assemblies, RcaT directly contacts the msDNA methylation site (a). Methylation promotes complex disassembly and partial release of RcaT (b).

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Panos Spatharas @pspatharas.bsky.social · 14/10/2026

3/11 Our cryo-EM structure reveals RcaT encased as an inactive dimer within a four-component core retron complex that can further assemble into filaments.

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Panos Spatharas @pspatharas.bsky.social · 14/10/2026

2/11 Retrons produce multicopy single-stranded DNA (msDNA) to monitor DNA-targeting phage proteins during infection. While activation by msDNA degradation is easier to envision, how subtle chemical modifications such as methylation activate retrons has remained enigmatic.

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Panos Spatharas @pspatharas.bsky.social · 14/10/2026

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Panos Spatharas @pspatharas.bsky.social · 14/10/2026

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Panos Spatharas @pspatharas.bsky.social · 14/10/2026

7/11 Together, our findings support a model in which methylation promotes msDNA displacement, exposing the previously occluded dimer–dimer interface. This allows RcaT tetramerisation and activation, coupled to retron complex disassembly.

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Panos Spatharas @pspatharas.bsky.social · 14/10/2026

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Panos Spatharas @pspatharas.bsky.social · 14/10/2026

5/11 We next examined RcaT after release. It forms tetramers consisting of two stacked dimers, with additional dimers stacking to form higher-order oligomers.

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Panos Spatharas @pspatharas.bsky.social · 14/10/2026

4/11 In these assemblies, RcaT directly contacts the msDNA methylation site (a). Methylation promotes complex disassembly and partial release of RcaT (b).

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Panos Spatharas @pspatharas.bsky.social · 14/10/2026

3/11 Our cryo-EM structure reveals RcaT encased as an inactive dimer within a four-component core retron complex that can further assemble into filaments.

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Panos Spatharas @pspatharas.bsky.social · 14/10/2026

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