RESUMEN
Ubiquitination of proliferating cell nuclear antigen (PCNA) at lysine 164 (K164) activates DNA damage tolerance pathways. Currently, we lack a comprehensive understanding of how PCNA K164 ubiquitination promotes genome stability. To evaluate this, we generated stable cell lines expressing PCNAK164R from the endogenous PCNA locus. Our data reveal that the inability to ubiquitinate K164 causes perturbations in global DNA replication. Persistent replication stress generates under-replicated regions and is exacerbated by the DNA polymerase inhibitor aphidicolin. We show that these phenotypes are due, in part, to impaired Fanconi anemia group D2 protein (FANCD2)-dependent mitotic DNA synthesis (MiDAS) in PCNAK164R cells. FANCD2 mono-ubiquitination is significantly reduced in PCNAK164R mutants, leading to reduced chromatin association and foci formation, both prerequisites for FANCD2-dependent MiDAS. Furthermore, K164 ubiquitination coordinates direct PCNA/FANCD2 colocalization in mitotic nuclei. Here, we show that PCNA K164 ubiquitination maintains human genome stability by promoting FANCD2-dependent MiDAS to prevent the accumulation of under-replicated DNA.
Asunto(s)
Reparación del ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi , Humanos , ADN/metabolismo , Daño del ADN , Replicación del ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Inestabilidad Genómica , Antígeno Nuclear de Célula en Proliferación/metabolismo , UbiquitinaciónRESUMEN
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.