Cervantes and Quijote protect heterochromatin from aberrant recombination and lead the way to the nuclear periphery.

Repairing double-strand breaks (DSBs) is particularly challenging in heterochromatin, where the abundance of repeated sequences exacerbates the risk of ectopic recombination and chromosome rearrangements. In Drosophila cells, faithful homologous recombination (HR) repair of heterochromatic DSBs relies on a specialized pathway that relocalizes repair sites to the nuclear periphery before Rad51 recruitment. Here we show that HR progression is initially blocked inside the heterochromatin domain by SUMOylation and the coordinated activity of two distinct Nse2 SUMO E3 ligases: Quijote (Qjt) and Cervantes (Cerv). In addition, the SUMO-targeted ubiquitin ligase (STUbL) Dgrn, but not its partner dRad60, is recruited to heterochromatic DSBs at early stages of repair and mediates relocalization. However, Dgrn is not required to prevent Rad51 recruitment inside the heterochromatin domain, suggesting that the block to HR progression inside the domain and relocalization to the nuclear periphery are genetically separable pathways. Further, SUMOylation defects affect relocalization without blocking heterochromatin expansion, revealing that expansion is not required for relocalization. Finally, nuclear pores and inner nuclear membrane proteins (INMPs) anchor STUbL/RENi components and repair sites to the nuclear periphery, where repair continues. Together, these studies reveal a critical role of SUMOylation and nuclear architecture in the spatial and temporal regulation of heterochromatin repair and the protection of genome integrity.