GNL3 SUMOylation is essential for DNA double-strand break repair by homologous recombination.

DNA double-strand break repair via homologous recombination is critical for maintaining genomic integrity and requires proper DNA end resection. Here, we identify GNL3, a nucleolar GTP-binding protein, as a key regulator of homologous recombination in human cells via SUMOylation-dependent control of DNA end resection. Expression of wild-type GNL3, but not the SUMO-defective K196R mutant, abolished DNA damage induced by knockdown of endogenous GNL3. GNL3 interacts with the BLM-DNA2 helicase-nuclease complex, promoting DNA end resection and subsequent RPA and RAD51 loading. This interaction requires SUMOylation and SUMO-interacting motifs in both proteins. We further show that USP36 acts as a SUMO ligase for GNL3, while SENP3 deSUMOylates GNL3. Breast cancer-derived GNL3 variants disrupting its SUMOylation or SUMO-interacting motif fail to interact with the BLM-DNA2 complex. GNL3 depletion sensitizes homologous recombination-proficient breast cancer cells to etoposide and Olaparib treatment, highlighting GNL3 and its SUMOylation as potential therapeutic targets in cancer.