Abstract
Sumoylation is an important enhancer of responses to DNA replication stress and the SUMO-targeted ubiquitin E3 ligase RNF4 regulates these responses by ubiquitylation of sumoylated DNA damage response factors. The specific targets and functional consequences of RNF4 regulation in response to replication stress, however, have not been fully characterized. Here we demonstrated that RNF4 is required for the restart of DNA replication following prolonged hydroxyurea (HU)-induced replication stress. Contrary to its role in repair of γ-irradiation-induced DNA double-strand breaks (DSBs), our analysis revealed that RNF4 does not significantly impact recognition or repair of replication stress-associated DSBs. Rather, using DNA fiber assays, we found that the firing of new DNA replication origins, which is required for replication restart following prolonged stress, was inhibited in cells depleted of RNF4. We also provided evidence that RNF4 recognizes and ubiquitylates sumoylated Bloom syndrome DNA helicase BLM and thereby promotes its proteosome-mediated turnover at damaged DNA replication forks. Consistent with it being a functionally important RNF4 substrate, co-depletion of BLM rescued defects in the firing of new replication origins observed in cells depleted of RNF4 alone. We concluded that RNF4 acts to remove sumoylated BLM from collapsed DNA replication forks, which is required to facilitate normal resumption of DNA synthesis after prolonged replication fork stalling and collapse.
Highlights
Accurate DNA replication is essential to maintenance of genome integrity
These observations suggested that RNF4 plays a unique role in the response to DNA replication stress, and that this role involves functions at sites of collapsed replication forks that may be independent of double-strand breaks (DSBs) repair
Extending the results of earlier proteomic studies (Kumar et al, 2017), we showed that BLM interacts with RNF4, sumoylated BLM is ubiquitylated by RNF4 in vitro, and RNF4 depletion led to a substantial increase in SUMO-BLM in HU treated cells (Figure 5)
Summary
Accurate DNA replication is essential to maintenance of genome integrity. When the replicative polymerase encounters DNA damage such as chemical modifications of bases, the polymerase stalls at the site of the DNA lesion and the CDC45-MCM2-7-GINS (CMG) helicase uncouples from the polymerase and continues to unwind downstream duplex to expose single-stranded DNA (ssDNA) (Cortez, 2019). ssDNA binding protein (RPA) binds to ssDNA, and the complex activates the ATR kinase, which is required for the recruitment of factors from the homologous recombination (HR) pathway (Dungrawala et al, 2015). SsDNA binding protein (RPA) binds to ssDNA, and the complex activates the ATR kinase, which is required for the recruitment of factors from the homologous recombination (HR) pathway (Dungrawala et al, 2015) These factors stabilize and protect the replication fork from nascent-strand degradation (Schlacher et al, 2011). Uncoupling occurs due to DNA polymerases stalling on the template following a >50% decrease in purine deoxynucleotide concentration (Skoog and Bjursell, 1974). In this context, activation of ATR and the recruitment of HR factors, such as RAD51 and BRCA2, are replication-specific (Petermann et al, 2010; Zellweger et al, 2015). The majority of dormant origins fired in these cells are >100 kb away from the collapsed forks
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