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 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.

The nucleolar protein GNL3 prevents resection of stalled replication forks

“…We reported previously our use of the iPOND method to identify novel factors associated with replication forks (Ribeyre et al, 2021). 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 (Figure 1A).…”

“…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 (Figure 1A). Those proteins that were significantly enriched in the pulse-labelled samples when compared to the chase were defined as components of the replisome (Ribeyre et al, 2021). These components included many proteins that were not previously known to be associated with nascent DNA.…”

“…B . Bar plot showing the LogRatio (pulse/chase) of average peptides intensities corresponding to the indicated proteins from (Ribeyre et al, 2021). Pulse experiments have been repeated 6 times and chase experiments 4 times.…”

“…Using the iPOND–MS data (Ribeyre et al, 2021), we calculated the logRatio of GNL3 in the pulse and the chase samples and found that it had a similar profile than known replisome components such as PCNA, RFC1 and FEN1 (Figure 1B). Also, by western blotting the iPOND proteins, we observed that GNL3, like PCNA, was associated with EdU only when the Click reaction was performed and was not found in the chase (Figure 1C), further supporting the conclusion that it is a replisome component.…”

“…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 (Lossaint et al, 2013; Ribeyre et al, 2021; 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.…”