FANCD2-dependent mitotic DNA synthesis relies on PCNA K164 ubiquitination

• Published in: Cell reports (Cambridge) Vol. 42; no. 12; p. 113523
• Main Authors: Leung, Wendy, Baxley, Ryan M., Traband, Emma, Chang, Ya-Chu, Rogers, Colette B., Wang, Liangjun, Durrett, Wesley, Bromley, Kendall S., Fiedorowicz, Lidia, Thakar, Tanay, Tella, Anika, Sobeck, Alexandra, Hendrickson, Eric A., Moldovan, George-Lucian, Shima, Naoko, Bielinsky, Anja-Katrin
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 26-12-2023; Elsevier
• Subjects: CP: Molecular biology; DNA - metabolism; DNA Damage; DNA Repair; DNA Replication; FANCD2; Fanconi Anemia Complementation Group D2 Protein - genetics; Fanconi Anemia Complementation Group D2 Protein - metabolism; Genomic Instability; Humans; MiDAS; PCNA; Proliferating Cell Nuclear Antigen - metabolism; replication stress; Ubiquitination; CP: Molecular biology; replication stress; PCNA; FANCD2; ubiquitination; MiDAS
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2023.113523

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.
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•PCNA K164 ubiquitination promotes mitotic DNA synthesis in non-transformed human cells•Robust FANCD2 ubiquitination, chromatin association, and foci formation rely on PCNA K164•PCNA and FANCD2 association in G2/M-phase nuclei requires PCNA ubiquitination at K164
Leung et al. show that FANCD2-dependent mitotic DNA synthesis (MiDAS) in non-transformed human cells relies on PCNA lysine 164 (K164) ubiquitination. Specifically, PCNA K164 ubiquitination promotes FANCD2 ubiquitination and recruitment to under-replicated regions and coordinates interactions in mitotic nuclei to promote MiDAS.