RNF4-mediated polyubiquitination regulates the Fanconi anemia/BRCA pathway

• Published in: The Journal of clinical investigation Vol. 125; no. 4; pp. 1523 - 1532
• Main Authors: Xie, Jenny, Kim, Hyungjin, Moreau, Lisa A., Puhalla, Shannon, Garber, Judy, Al Abo, Muthana, Takeda, Shunichi, D’Andrea, Alan D.
• Format: Journal Article
• Language: English
• Published: United States American Society for Clinical Investigation 01.04.2015
• Subjects: Adult; Binding sites; Biomedical research; BRCA1 Protein - physiology; Cell Line, Tumor; Deoxyribonucleic acid; Development and progression; DNA; DNA repair; DNA Repair - genetics; DNA Repair - physiology; Fanconi Anemia - genetics; Fanconi Anemia Complementation Group A Protein - genetics; Fanconi Anemia Complementation Group A Protein - physiology; Fanconi Anemia Complementation Group Proteins - antagonists & inhibitors; Fanconi Anemia Complementation Group Proteins - genetics; Fanconi Anemia Complementation Group Proteins - physiology; Fanconi's anemia; Female; Genes, BRCA1; Genetic aspects; Health aspects; Humans; Laboratories; Mutation; Mutation, Missense; Nuclear Proteins - physiology; Pedigree; Phenotype; Plasmids; Point Mutation; Proteasome Endopeptidase Complex - metabolism; Protein Binding; Protein Processing, Post-Translational - physiology; Proteins; Proteolysis; RNA, Small Interfering - pharmacology; Signal Transduction - physiology; Sumoylation; Transcription factors; Transcription Factors - physiology; Triple Negative Breast Neoplasms - genetics; Ubiquitin-proteasome system; Ubiquitination - physiology
• ISSN: 0021-9738; 1558-8238; 1558-8238
• DOI: 10.1172/JCI79325

The Fanconi anemia/BRCA (FA/BRCA) pathway is a DNA repair pathway that is required for excision of DNA interstrand cross-links. The 17 known FA proteins, along with several FA-associated proteins (FAAPs), cooperate in this pathway to detect, unhook, and excise DNA cross-links and to subsequently repair the double-strand breaks generated in the process. In the current study, we identified a patient with FA with a point mutation in FANCA, which encodes a mutant FANCA protein (FANCAI939S). FANCAI939S failed to bind to the FAAP20 subunit of the FA core complex, leading to decreased stability. Loss of FAAP20 binding exposed a SUMOylation site on FANCA at amino acid residue K921, resulting in E2 SUMO-conjugating enzyme UBC9-mediated SUMOylation, RING finger protein 4-mediated (RNF4-mediated) polyubiquitination, and proteasome-mediated degradation of FANCA. Mutation of the SUMOylation site of FANCA rescued the expression of the mutant protein. Wild-type FANCA was also subject to SUMOylation, RNF4-mediated polyubiquitination, and degradation, suggesting that regulated release of FAAP20 from FANCA is a critical step in the normal FA pathway. Consistent with this model, cells lacking RNF4 exhibited interstrand cross-linker hypersensitivity, and the gene encoding RNF4 was epistatic with the other genes encoding members of the FA/BRCA pathway. Together, the results from our study underscore the importance of analyzing unique patient-derived mutations for dissecting complex DNA repair processes.