RNF4 and USP7 cooperate in ubiquitin-regulated steps of DNA replication

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Abstract
DNA replication requires precise regulation achieved through post-translational modifications, including ubiquitination and SUMOylation. These modifications are linked by the SUMO-targeted E3 ubiquitin ligases (STUbLs). Ring finger protein 4 (RNF4), one of only two mammalian STUbLs, participates in double-strand break repair and resolving DNA–protein cross-links. However, its role in DNA replication has been poorly understood. Using CRISPR/Cas9 genetic screens, we discovered an unexpected dependency of RNF4 mutants on ubiquitin specific peptidase 7 ( USP7) for survival in TP53 -null retinal pigment epithelial cells. TP53 −/– /RNF4 −/– /USP7 −/– triple knockout (TKO) cells displayed defects in DNA replication that cause genomic instability. These defects were exacerbated by the proteasome inhibitor bortezomib, which limited the nuclear ubiquitin pool. A shortage of free ubiquitin suppressed the ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint response, leading to increased cell death. In conclusion, RNF4 and USP7 work cooperatively to sustain a functional level of nuclear ubiquitin to maintain the integrity of the genome.
Funding Information
  • National Science Foundation (MCB1818293)
  • Minnesota Clinical and Translational Science Institute (UL1TR002493, UL1TR002494)
  • National Institute of Health (5F30CA257227-02, R01GM074917, R35GM141805)

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