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Faithful DNA replication requires specific proteins that protect replication forks and so prevent the formation of DNA lesions that may damage the genome. Identification of new proteins involved in this process is essential to understand how DNA lesions accumulate in cancer cells and how they tolerate them. Here, we show that human GNL3/nucleostemin, a GTP‐binding protein localized mostly in the nucleolus and highly expressed in cancer cells, prevents nuclease‐dependent resection of nascent DNA in response to replication stress. We demonstrate that inhibiting origin firing reduces resection. This suggests that the heightened replication origin activation observed upon GNL3 depletion largely drives the observed DNA resection probably due to the exhaustion of the available RPA pool. We show that GNL3 and DNA replication initiation factor ORC2 interact in the nucleolus and that the concentration of GNL3 in the nucleolus is required to limit DNA resection. We propose that the control of origin firing by GNL3 through the sequestration of ORC2 in the nucleolus is critical to prevent nascent DNA resection in response to replication stress. Synopsis: GNL3 limits the number of fired replication origins by sequestering ORC2 within the nucleolus. In the absence of GNL3, increased replication origin firing triggers nuclease‐mediated resection of newly synthesized DNA in response to replication stress.GNL3 is a GTP‐binding protein primarily found within the nucleolus.The nucleolar accumulation of GNL3 limits the number of replication origins that fire, likely through the sequestration of ORC2.When GNL3 is deficient or fails to accumulate in the nucleolus, it triggers nuclease‐mediated resection of newly synthesized DNA in response to replication stress. [ABSTRACT FROM AUTHOR]

Coordination between transcription and replication is crucial in the maintenance of genome integrity. Disturbance of these processes leads to accumulation of aberrant DNA: RNA hybrids (R-loops) that, if unresolved, generate DNA damage and genomic instability. Here we report a novel, unexpected role for the nucleopore-associated mRNA export factor Ddx19 in removing nuclear R-loops formed upon replication stress or DNA damage. We show, in live cells, that Ddx19 transiently relocalizes from the nucleopore to the nucleus upon DNA damage, in an ATR/Chk1-dependent manner, and that Ddx19 nuclear relocalization is required to clear R-loops. Ddx19 depletion induces R-loop accumulation, proliferation-dependent DNA damage and defects in replication fork progression. Further, we show that Ddx19 resolves R-loops in vitro via its helicase activity. Furthermore, mutation of a residue phosphorylated by Chk1 in Ddx19 disrupts its interaction with Nup214 and allows its nuclear relocalization. Finally, we show that Ddx19 operates in resolving R-loops independently of the RNA helicase senataxin. Altogether these observations put forward a novel, ATR-dependent function for Ddx19 in R-loop metabolism to preserve genome integrity in mammalian cells. [ABSTRACT FROM AUTHOR]