GNL3 regulates replication origin firing and protects stalled replication forks

DNA replication by the replisome requires specific proteins that protect replication forks and so prevent the formation of DNA lesions that may damage the genome. Here, we show that human GNL3/nucleostemin, a GTP-binding protein localized in the nucleolus and the nucleoplasm, is a new component of the replisome. Depletion of GNL3 reduces fork speed but increases replication origin firing. When subjected to replication stress, the nascent DNA of GNL3-depleted cells undergoes nuclease-dependent resection, a source of DNA lesions. Inhibition of origin firing decreases this resection, indicating that the increased replication origin firing seen upon GNL3 depletion mainly accounts for the observed DNA resection. We show that GNL3 and DNA replication initiation factor ORC2 interact in the nucleolus, and that GNL3 regulates ORC2 subnuclear localization. The accumulation of GNL3 in the nucleolus is thus required to limit DNA resection in response to replicative stress, potentially through the regulation of ORC2 functions.