Eukaryotic genomes are duplicated from thousands of replication origins that fire sequentially forming a defined spatiotemporal pattern of replication clusters. The temporal order of DNA replication is determined by chromatin architecture and, more specifically, by chromatin contacts that are stabilized by RIF1. Here, we show that RIF1 localizes near newly synthesized DNA. In cells exposed to the DNA replication inhibitor aphidicolin, suppression of RIF1 markedly decreased the efficacy of isolation of proteins on nascent DNA, suggesting that the isolation of proteins on nascent DNA procedure is biased by chromatin topology. RIF1 was required to limit the accumulation of DNA lesions induced by aphidicolin treatment and promoted the recruitment of cohesins in the vicinity of nascent DNA. Collectively, the data suggest that the stabilization of chromatin topology by RIF1 limits replication-associated genomic instability.

Eukaryotic genomes are duplicated from thousands of replication origins that fire sequentially forming a defined spatiotemporal pattern of replication clusters. The importance of the organization of replisomes into functional clusters, also called replication factories, is still poorly understood. Here we identified the multifunctional protein RIF1 as a structural component of replication factories. RIF1 depletion did not impair the velocity of replication forks, neither in basic conditions nor in presence of a molecule that interferes with replication fork progression but increased the frequency of DNA lesions induced in S phase. Isolation of replication-associated proteins from RIF1-depleted cells revealed a major defect in the clustering of replication factors on nascent DNA, without any noticeable impact on DNA synthesis or replisome stability. We found that the changes in replication patterns commonly observed upon RIF1 depletion are induced by DNA replication stress. The data suggest that RIF1 encases replication factories to ensure the organization of replication clusters against chromatin rearrangements induced by DNA replication stress.

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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.