Alessandro Carlino1,2*, Bryce Collison1,2, Mia Bellner1, Eleonora De Vitis1, Tam Quach1, and Ilaria Palmisano1

Transient DNA double‑strand breaks (DSBs) arise in neurons during intense transcriptional activity to relieve torsional stress. Although physiologically important, their persistence or inefficient repair threatens genomic stability, making tight control of their formation and repair essential. In the peripheral nervous system, temporary DSBs accompany the activation of regeneration‑associated genes after nerve injury, yet how these breaks are repaired remains unclear. We recently showed that the cohesin complex, a key regulator of genome architecture, is required for activating regenerative genes in mouse dorsal root ganglia (DRG) neurons after sciatic nerve injury. Cohesin organizes the three‑dimensional chromatin around these loci, enabling their injury‑induced transcription. Beyond this architectural role, cohesin has been implicated in DNA repair in dividing cells, raising the question of whether it also regulates DSB repair in post‑mitotic neurons. To test this, we quantified DSB levels by immunohistochemical analysis in wild‑type and cohesin‑depleted DRG neurons three days after sciatic nerve crush. Cohesin‑depleted neurons displayed elevated DSB levels, indicating impaired repair of injury‑induced breaks. Using Etoposide (ETP), a Topoisomerase II inhibitor that generates persistent DSBs, we further observed robust recruitment of cohesin to DSB foci in cultured neurons and in vivo, and colocalization with 53BP1, a canonical repair effector. To assess whether cohesin is required for 53BP1 recruitment, we depleted the core subunit SMC3 by siRNA nucleofection. Cohesin depletion resulted in decreased 53BP1 levels at ETP-induced DSB foci. Transcriptomic analysis of DRG neurons from cohesin‑deficient mice after nerve injury revealed strong upregulation of inflammatory, cGAS–STING, interferon, and senescence pathways, consistent with persistent DNA damage. Together, these findings suggest that cohesin coordinates the repair of transcription‑associated DSBs during nerve regeneration by enabling the recruitment of DNA repair factors to damage sites. Together, these findings suggest a role for cohesin in coordinating the repair of transcription-associated DSBs during nerve regeneration.