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

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.

Automated summary

DNA replication is regulated by post-translational modifications, including ubiquitination and SUMOylation, which are linked by the SUMO-targeted E3 ubiquitin ligases (STUbLs). RNF4, one of the mammalian STUbLs, is involved in DNA repair, but its role in replication was unclear. Through genetic screens, RNF4 mutants were found to depend on USP7 for survival in TP53-null cells. TKO cells displayed replication defects and increased cell death due to limited nuclear ubiquitin caused by proteasome inhibition.