The organizer of chromatin topology RIF1 ensures cellular resilience to DNA replication stress

Abstract

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.

The nucleolar protein GNL3 prevents resection of stalled replication forks

“…Therefore, the weak association of GNL3 to nascent DNA may simply reflect its localization in proximity of heterochromatic regions undergoing DNA replication. This is supported by our recent findings showing that iPOND efficacy is biased by genome organization (Lebdy et al, 2023). Based on this we propose that the slow replication fork speed observed upon GNL3 depletion is more likely a compensation mechanism of the increased replication origin firing (Ge et al, 2007; Ibarra et al, 2008) rather than a direct impact on replication forks speed.…”

“…We reported previously our use of the iPOND method to identify novel factors associated with replication forks (Lebdy et al, 2023). Briefly, we pulse-labelled newly synthesized DNA in Hela S3 cells with 5-ethynyl-2’-deoxyuridine (EdU, a nucleoside analogue of thymidine that can be labelled by Click chemistry) or pulsed with EdU then chased for two hours with thymidine, then we purified the proteins associated with EdU.…”

“…Briefly, we pulse-labelled newly synthesized DNA in Hela S3 cells with 5-ethynyl-2’-deoxyuridine (EdU, a nucleoside analogue of thymidine that can be labelled by Click chemistry) or pulsed with EdU then chased for two hours with thymidine, then we purified the proteins associated with EdU. Those proteins that were significantly enriched in the pulse-labelled samples when compared to the chase were defined as components of the replisome (Lebdy et al, 2023). These components included many proteins that were not previously known to be associated with nascent DNA.…”

“…In addition, the reduced fork velocity observed upon GNL3 depletion may also impair the replication of specific regions. We detected GNL3 in vicinity of replication forks using iPOND coupled with mass spectrometry (Lebdy et al, 2023) and Western-blot (Fig 1F) suggesting that its depletion may directly impairs fork speed. Yet, the recruitment of GNL3 at replication forks is low and it was not detected by other iPOND screens performed in basal conditions (Lopez-Contreras et al, 2013; Sirbu et al, 2013).…”

“…We previously used the iPOND (isolation of proteins on nascent DNA) method coupled with mass spectrometry (iPOND-MS) to identify novel factors associated with replication forks (Lebdy et al, 2023; Lossaint et al, 2013; Ribeyre et al, 2016). Here, we used an siRNA screen to identify those novel factors whose depletion increases the number of DNA lesions in response to replication stress.…”