The Fanconi Anemia DNA Repair Pathway Is Regulated by an Interaction between Ubiquitin and the E2-like Fold Domain of FANCL.
The ELF domain is found in all FANCL homologues, yet the function of the domain remains unknown.We show that the interaction is not necessary for the recognition of the core complex, it does not enhance the interaction between FANCL and Ube2T, and is not required for FANCD2 monoubiquitination in vitro.However, we demonstrate that the ELF domain is required to promote efficient DNA damage-induced FANCD2 monoubiquitination in vertebrate cells, suggesting an important function of ubiquitin binding by FANCL in vivo.
Affiliation: From the Protein Structure and Function Laboratory, Lincoln's Inn Fields Laboratories of the London Research Institute, Cancer Research, United Kingdom, 44 Lincoln's Inn Fields, London WC2A 3LY, United Kingdom.
- DNA Repair*
- Drosophila Proteins/chemistry*/genetics/metabolism
- Fanconi Anemia Complementation Group L Protein/chemistry*/genetics/metabolism
- Fanconi Anemia Complementation Group Proteins/chemistry*/genetics/metabolism
- Xenopus Proteins/chemistry*/genetics/metabolism
- Amino Acid Sequence
- Binding Sites
- Drosophila melanogaster
- Fanconi Anemia/genetics
- Gene Expression Regulation
- Models, Molecular
- Molecular Sequence Data
- Protein Binding
- Protein Folding
- Protein Interaction Domains and Motifs
- Protein Structure, Secondary
- Recombinant Proteins/chemistry/genetics/metabolism
- Sequence Alignment
- Signal Transduction
- Ubiquitin-Conjugating Enzymes/genetics/metabolism
- Xenopus laevis
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Figure 8: Ubiquitin binding by FANCL is required for efficient FANCD2/FANCI monoubiquitination in vertebrate cells.A, FANCL-deficient DT40 cells (fancl−/−) were complemented with TAP-tagged wild-type FANCL (TAP-FANCL) and FANCL with mutated ELF ubiquitin-binding sites (TAP-FANCL(L7A, L79A), TAP-FANCL(L7A, D78A, L79A, V80A), TAP-FANCL(L7A, D78R, L79A)). Cells were either 150 nm MMC-treated (+) or mock treated (−), and lysates were subfractionated into high salt nuclear extract (NEX) and soluble chromatin extract (CHEX). Equal total protein amount of extracts were separated on SDS-PAGE gels and analyzed by immunoblotting using anti-FANCD2 and anti-TAP antibodies. Mutations in the ELF ubiquitin-binding site perturbed MMC-induced FANCD2 monoubiquitination. D2-Ub, monoubiquitinated FANCD2; D2, unmodified FANCD2. B, quantitation of the various ratios of monoubiquitinated FANCD2 and unmodified FANCD2 shown in A using ImageJ analysis software. Standard error of the mean is given from three independent experiments. C, cell lines described in A were exposed to 150 ng/ml MMC, whole cell extract prepared after indicated times and subjected to FANCD2 immunoblot analysis. D, quantitation of the various ratios of monoubiquitinated FANCD2 and unmodified FANCD2 shown in C using ImageJ analysis software. D, indicated cell lines were treated with 600 nm MMC (+) or mock treated (−), fractionated as described in A, and analyzed by immunoplotting using anti-FANCI and anti-TAP. FANCI monoubiquitination was significantly reduced in ELF-mutated cells. I-Ub, monoubiquitinated FANCI; I, unmodified FANCI. F, TAP-tagged wild-type FANCL (TAP-FANCL) and ELF-mutated FANCL (TAP-FANCL [L7A, D78R, L79A]) were affinity-purified from corresponding DT40 cells with IgG-Sepharose, and incubated with either wild type HA-ubiquitin (WT) or I44A mutated HA-ubiquitin (I44A). Co-precipitation of the ubiquitin forms were analyzed by immunoplotting using anti-HA. Mutating the ELF domain or the ubiquitin I44 hydrophobic patch disrupted the TAP-FANCL ubiquitin interaction. G, TAP-FANCL and TAP-FANCL (L7A D78R L79A) high salt nuclear extracts were fractionated by Superose 6 size exclusion chromatography, and fractions were analyzed by immunoplotted using anti-TAP. Elution profiles of a 1–1.5 MDa complex were comparable between wild type FANCL and the ELF domain mutated FANCL.
Finally, we asked whether ubiquitin binding by FANCL has any relevance to FANCD2 monoubiquitination in cells. To test this hypothesis we expressed ELF domain-mutated versions of TAP-tagged FANCL in FANCL-deficient avian DT40 cells (fancl−/−). These FANCL variants carry combinatorial point mutations of conserved amino acids, L7A, D78A, D78R, L79A, V80A, that assemble the ubiquitin binding surface determined from Drosophila FANCL (Fig. 6A). Wild-type TAP-tagged FANCL expression promotes efficient FANCD2 monoubiquitination both in high salt nuclear extracts (NEX) and soluble chromatin extracts (CHEX) (Fig. 8). In contrast, TAP-FANCL (L7A, L79A), TAP-FANCL (L7A, D78A, L79A, V80A), and TAP-FANCL (L7A, D78R, L79A) are defective in mitomycin C (MMC)-induced monoubiquitination of FANCD2 (Fig. 8, A and B). In addition we compared time-dependent FANCD2 monoubiquitination following MMC treatment of wild-type TAP-FANCL with ELF domain mutants FANCD2 monoubiquitination following MMC treatment of wild type TAP-FANCL with ELF ubiquitin binding mutants TAP-FANCL (L7A, L79A), TAP-FANCL (L7A, D78A, L79A, V80A), and TAP-FANCL (L7A, D78R, L79A) (Fig. 8, C and D). TAP-FANCL (L7A, D78A, L79A, V80A), TAP-FANCL (L7A, D78R, L79A), and to a lesser extent TAP-FANCL (L7A, L79A), showed a significant delay in and overall reduction of FANCD2 monoubiquitination. Moreover, we observed a similar reduction of MMC-induced FANCI monoubiquitination (Fig. 8E).