RNF4 Regulates the BLM Helicase in Recovery From Replication Fork Collapse

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.

Replication Stress: A Review of Novel Targets to Enhance Radiosensitivity-From Bench to Clinic

“…It also promotes DNA-end resection, restart of stalled replication forks, and processing of ultrafine DNA bridges in mitosis (111). BLM helicase-deficient cells exhibit multiple defects in DNA replication, including accumulation of abnormal DNA replication intermediates, slower replication fork velocity, and excessive firing of dormant origins, thus exhibit increased levels of chromatid breakage and HR (112). It interacts directly with both RAD51 and RPA, and the function in DNA replication is regulated by sumoylation (113).…”

BLM Sumoylation Is Required for Replication Stability and Normal Fork Velocity During DNA Replication

“…Consequently, we might expect that inhibition of the processing of SUMO-BLM by the STUbL RNF4 by siRNA depletion might produce some similar phenotypes by leaving an excess of immobile, albeit sumoylated, BLM protein at sites of damage. We recently reported that SUMO-BLM is a substrate of RNF4 and RNF4 depletion led to a hyper-accumulation of SUMO-BLM at collapsed forks generated by prolonged HU treatment (Ellis et al, 2021). Consequently, we expected that sumoylated BLM would also be less mobile at collapsed forks.…”

“…In a careful examination of other phenotypes, we noted little phenotypic concordance between SM-BLM cells and RNF4deficient cells. The differences are enumerated as follows (Ouyang et al, 2009;Ouyang et al, 2013;Ellis et al, 2021): 1) Numbers of BLM foci in untreated SM-BLM cells were 2.5 times higher but these numbers were not increased after treatment with HU, whereas BLM foci in untreated RNF4-deficient cells were no different from untreated control and HU treatment increased the median number by over 20%. 2) γ-H2AX focal numbers were significantly elevated in untreated SM-BLM cells, and they increased excessively after HU treatment, but the numbers of γ-H2AX foci were not different from control in untreated or HUtreated RNF4-deficient cells.…”

“…Once recruited to the SMC5/6 complex, BLM is sumoylated by the NSMCE2 SUMO E3 ligase component of that complex (Pond et al, 2019). We have shown previously that SUMO-BLM is a substrate for SUMO- targeted ubiquitin ligase (STUbL) RNF4, suggesting that sumoylation may be required for BLM turnover at stalled and collapsed forks (Ellis et al, 2021). Consequently, we asked whether BLM dynamics at collapsed replication forks were affected by sumoylation, by performing fluorescence recovery after photobleaching (FRAP) at sites of replication collapse in BLM+ and SM-BLM cells generated by treatment with 2 mM HU for 24 h (Figure 3).…”

SUMO enhances unfolding of SUMO–polyubiquitin-modified substrates by the Ufd1/Npl4/Cdc48 complex

“…These priming conjugation events can be further remodeled by ligases and proteases to generate ubiquitin and ubiquitin-like polymeric chains with different topologies ( 4 ), each of which has the potential to signal through factors that recognize different chain topologies. Several studies suggest overlap in ubiquitin and SUMO conjugation pathways in various cellular processes including heat shock and DNA damage responses and the maintenance of subcellular structures including promyelocytic leukemia or PML bodies ( 5 – 11 ).…”