RNF4-mediated polyubiquitination regulates the Fanconi anemia/BRCA pathway
Jenny Xie, … , Shunichi Takeda, Alan D. D’Andrea
Jenny Xie, … , Shunichi Takeda, Alan D. D’Andrea
Published March 9, 2015
Citation Information: J Clin Invest. 2015;125(4):1523-1532. https://doi.org/10.1172/JCI79325.
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Research Article Oncology

RNF4-mediated polyubiquitination regulates the Fanconi anemia/BRCA pathway

Abstract

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.

Authors

Jenny Xie, Hyungjin Kim, Lisa A. Moreau, Shannon Puhalla, Judy Garber, Muthana Al Abo, Shunichi Takeda, Alan D. D’Andrea

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Figure 3

The full-length mutant FANCA protein in DF2231 cells is unstable and has enhanced SUMO and ubiquitin conjugation.

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The full-length mutant FANCA protein in DF2231 cells is unstable and has...
(A) Relative survival of wild-type human GM0637 cells, FA-I cells, or DF2231 cells derived from a patient with FA-A treated with increasing doses of MMC and plated for 5 days. Three independent experiments were performed. (B) Immunoblot analysis of wild-type (GM0637) or DF2231 fibroblasts treated with various DNA damage-inducing agents. (C) Immunoblot of FANCA, FANCG, and FAAP20 in lysates of DF2231 cells untreated or pretreated with 20 μM MG132 and GM0637 cells treated with siRNAs to control, FAAP20, or FANCG. (D) Endogenous FANCA was immunoprecipitated under denaturing conditions from cell lysates of FA-A (GM6914), wild-type (GM0637), or DF2231 cells, followed by anti-FANCA, -SUMO, or -ubiquitin immunoblot. (E) Endogenous FANCA was immunoprecipitated under denaturing conditions from cell lysates of wild-type (GM0637) or DF2231 cells transfected with siRNA oligos against control or UBC9, followed by anti-SUMO immunoblot. The thin black line indicates that noncontiguous lanes from the same blot are shown. (F) HeLa cells were transfected with HA-tagged SUMO3, and the cells were treated with UV, HU, or MMC for the indicated times. Endogenous FANCA SUMOylation was determined under denaturing conditions by anti-FANCA immunoprecipitation, followed by anti-FANCA or anti-HA immunoblot.