Structural basis for continuous DNA-end protection during ligation of double-strand breaks in yeast Non-Homologous End-Joining

missoury, s.; Tettaravou, C.; Castelli, S.; Pelletier, A.; Morin, V.; Varela, P. F.; Ropars, V.; Mattarocci, S.; Legrand, P.; Modesti, M.; Marcand, S.; Charbonnier, J.-B.; Delarue, M.

doi:10.64898/2026.03.12.711350

bioRxiv · 10.64898/2026.03.12.711350

Structural basis for continuous DNA-end protection during ligation of double-strand breaks in yeast Non-Homologous End-Joining

missoury, s.·Tettaravou, C.·Castelli, S.·Pelletier, A.·Morin, V.·Varela, P. F.·Ropars, V.·Mattarocci, S.·Legrand, P.·Modesti, M.·Marcand, S.·Charbonnier, J.-B.·Delarue, M.

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Abstract

Non-homologous end joining (NHEJ) repairs DNA double-strand breaks by synapsing and ligating DNA ends. In vertebrates, DNA-PKcs promotes end alignment and processing, yet several eukaryotes - including budding yeast - perform NHEJ without DNA-PKcs, and the underlying mechanism remains unclear. Here we report cryo-electron microscopy structures of reconstituted Saccharomyces cerevisiae NHEJ synaptic complexes assembled on DNA ends bearing either 4-bp terminal microhomologies or blunt termini. On the microhomology substrate with a single 5-P, we capture a ligation-competent short-range complex in which a single Dnl4 catalytic core is fully ordered and engaged at the 5'-adenylated nick. When two 5-P are present, we resolve two predominant, coexisting DNA-aligned protective states in which both Dnl4 DBD-NTD modules occupy the break region with reciprocal geometries, supporting an alternating engagement model for sequential sealing of the two strands while maintaining continuous end association. In contrast, blunt-ended substrates yield a non-aligned protective configuration in which two Dnl4 catalytic modules constrain the DNA ends [~]30 [A] apart in a ligation-incompatible arrangement, providing a structural explanation for slow blunt-end joining in yeast. Together, these structures define architectural and mechanistic principles of DNA-PKcs-independent NHEJ.

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missoury, s., Tettaravou, C., Castelli, S., Pelletier, A., Morin, V., Varela, P. F., Ropars, V., Mattarocci, S., Legrand, P., Modesti, M., Marcand, S., Charbonnier, J.-B., Delarue, M.. 2026-03-12. Structural basis for continuous DNA-end protection during ligation of double-strand breaks in yeast Non-Homologous End-Joining. https://doi.org/10.64898/2026.03.12.711350

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