Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Mar 7;289(10):7003-7010.
doi: 10.1074/jbc.M113.533976. Epub 2014 Jan 22.

The carboxyl terminus of FANCE recruits FANCD2 to the Fanconi Anemia (FA) E3 ligase complex to promote the FA DNA repair pathway

David Polito  1 , Scott Cukras  1 , Xiaozhe Wang  2 , Paige Spence  1 , Lisa Moreau  3 , Alan D D'Andrea  2 , Younghoon Kee  4
Affiliations

The carboxyl terminus of FANCE recruits FANCD2 to the Fanconi Anemia (FA) E3 ligase complex to promote the FA DNA repair pathway

David Polito et al. J Biol Chem. .

Abstract

Fanconi anemia (FA) is a genome instability syndrome characterized by bone marrow failure and cellular hypersensitivity to DNA cross-linking agents. In response to DNA damage, the FA pathway is activated through the cooperation of 16 FA proteins. A central player in the pathway is a multisubunit E3 ubiquitin ligase complex or the FA core complex, which monoubiquitinates its substrates FANCD2 and FANCI. FANCE, a subunit of the FA core complex, plays an essential role by promoting the integrity of the complex and by directly recognizing FANCD2. To delineate its role in substrate ubiquitination from the core complex assembly, we analyzed a series of mutations within FANCE. We report that a phenylalanine located at the highly conserved extreme C terminus, referred to as Phe-522, is a critical residue for mediating the monoubiquitination of the FANCD2-FANCI complex. Using the FANCE mutant that specifically disrupts the FANCE-FANCD2 interaction as a tool, we found that the interaction-deficient mutant conferred cellular sensitivity in reconstituted FANCE-deficient cells to a similar degree as FANCE null cells, suggesting the significance of the FANCE-FANCD2 interaction in promoting cisplatin resistance. Intriguingly, ectopic expression of the FANCE C terminus fragment alone in FA normal cells disrupts DNA repair, consolidating the importance of the FANCE-FANCD2 interaction in the DNA cross-link repair.

Keywords: DNA Damage; DNA Repair; E3 Ubiquitin Ligase; Protein Degradation; Protein Dynamics; Protein Folding; Protein Targeting; Protein-protein Interactions; Ubiquitin.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
The C terminus of FANCE is essential in promoting the FANCD2 monoubiquitination. MMC survival assays (A) and the FANCD2 monoubiquitination (Ub; B) for the CΔ25 mutant expressed in patient-derived FANCE-deficient fibroblasts (DF1179). An asterisk indicates a nonspecific band in the anti-FANCA blot. C, a summary of FANCE truncations and their ability to promote the FA pathway. D, anti-FLAG immunoprecipitation from the DF1179 lysate, followed by anti-FANCA, FANCC, FANCG Western blotting. E, left panel, sequence alignment of the FANCE C terminus. Right panel, solvent-accessible structure of the FANCE C terminus. The Protein Data Bank file was downloaded from RCSB Protein Data Bank code 2ILR. Residue Phe-522 is shown in red. F, analysis of FANCE point mutants within the C terminus for their ability to support FANCD2 monoubiquitination in the DF1179 FANCE-null fibroblasts. HU, hydroxylurea; R, resistant; S, sensitive; V, vector.
FIGURE 2.
FIGURE 2.
The FANCE Phe-522 residue is required for its interaction with FANCD2. A, yeast two-hybrid analysis of FANCD2 paired with FANCE and FANCF (see “Experimental Procedures”). B, immunoprecipitation of FLAG-FANCE WT and F522D from EUFA130 cells show that the Phe-522 residue is essential for the FA core complex to recognize an unmodified form of FANCD2. C, monoubiquitination (Ub) of FANCI is impaired in EUFA130 cells reconstituted with FLAG-FANCE FD mutant. Expression of the recombinant FANCE proteins (both WT and FD) is reduced upon hydroxylurea (HU) treatment, for unclear reason. D, yeast two-hybrid analysis of FANCE paired with FANCD2 and FANCI. Because the full-length DBD-FANCD2 did not yield GAL4 activation when coexpressed with AD-FANCI, the C terminus truncation containing residues 1–1196 was expressed as a DBD hybrid, in this experiment. It has been shown that the C terminus of FANCD2 is dispensable for interaction with FANCE (not shown).
FIGURE 3.
FIGURE 3.
FANCE F522D expressing cells are phenotypically similar to FANCE null cells. A, the F522D mutant EUFA130 cells elicit a normal DNA damage response. B, MMC survival assay. Chromosomal breakage analyses with representative images (C) and percentage of chromosomal aberrations per cell (D) in EUFA130 cells expressing either WT FANCE or the F522D mutant. These are based on three independent experiments. V, vector.
FIGURE 4.
FIGURE 4.
Expression of a C-terminal fragment of FANCE in FA-wild type cells disrupts the FA pathway. A, HeLa cells stably expressing pMSCV-FLAG-FANCE (273–536) WT, F522D, or empty vector (V) were treated with 2 mm hydroxylurea for 2, 6, or 24 h. Right panel, ImageJ software was used to quantify the monoubiquitination (Ub) status of FANCD2 as seen in the immunoblot. B, left panel, immunofluorescence of FANCD2 in the HeLa cell lines treated with 2 mm hydroxylurea (HU) for 24 h. Right panel, data obtained from counting ∼80 cells with at least 10 FANCD2 foci. C, cisplatin survival assay of HeLa cells stably expressing the corresponding FANCE fragments. D, a schematic model for the recruitment of the FANCD2-FANCI proteins to the FA core complex through the FANCE C terminus. The surface area of FANCE containing the Phe-522 residue is indicated as yellow oval. The determinant within the FANCD2-FANCI that interacts with FANCE is not well understood. PyMOL software was used for modeling the FANCD2-FANCI heterodimer (8).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Kee Y., D'Andrea A. D. (2012) Molecular pathogenesis and clinical management of Fanconi anemia. J. Clin. Investig. 122, 3799–3806 - PMC - PubMed
    1. Bogliolo M., Schuster B., Stoepker C., Derkunt B., Su Y., Raams A., Trujillo J. P., Minguillón J., Ramírez M. J., Pujol R., Casado J. A., Baños R., Rio P., Knies K., Zúñiga S., Benítez J., Bueren J. A., Jaspers N. G., Schärer O. D., de Winter J. P., Schindler D., Surrallés J. (2013) Mutations in ERCC4, encoding the DNA-repair endonuclease XPF, cause Fanconi anemia. Am. J. Hum. Genet. 92, 800–806 - PMC - PubMed
    1. Osorio A., Bogliolo M., Fernandez V., Barroso A., de la Hoya M., Caldes T., Lasa A., Ramon Y. C. T., Santamarina M., Vega A., Quiles F., Lazaro C., Diez O., Fernandez D., Gonzalez-Sarmiento R., Duran M., Piqueras J. F., Marin M., Pujol R., Surralles J., Benitez J. (2013) Evaluation of Rare Variants in the New Fanconi Anemia Gene ERCC4 (FANCQ) as Familial Breast/Ovarian Cancer Susceptibility Alleles. Hum. Mutat. 34, 1615–1618 - PubMed
    1. Kennedy R. D., D'Andrea A. D. (2005) The Fanconi Anemia/BRCA pathway: new faces in the crowd. Genes Dev. 19, 2925–2940 - PubMed
    1. Smogorzewska A., Matsuoka S., Vinciguerra P., McDonald E. R., 3rd, Hurov K. E., Luo J., Ballif B. A., Gygi S. P., Hofmann K., D'Andrea A. D., Elledge S. J. (2007) Identification of the FANCI protein, a monoubiquitinated FANCD2 paralog required for DNA repair. Cell 129, 289–301 - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources