Miles JA et al. (2015), The Fanconi Anemia DNA Repair Pathway Is Regula...

XB-ART-51604

J Biol Chem 2015 Aug 21;29034:20995-21006. doi: 10.1074/jbc.M115.675835.

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The Fanconi Anemia DNA Repair Pathway Is Regulated by an Interaction between Ubiquitin and the E2-like Fold Domain of FANCL.

Miles JA , Frost MG , Carroll E , Rowe ML , Howard MJ , Sidhu A , Chaugule VK , Alpi AF , Walden H .

Abstract
The Fanconi Anemia (FA) DNA repair pathway is essential for the recognition and repair of DNA interstrand crosslinks (ICL). Inefficient repair of these ICL can lead to leukemia and bone marrow failure. A critical step in the pathway is the monoubiquitination of FANCD2 by the RING E3 ligase FANCL. FANCL comprises 3 domains, a RING domain that interacts with E2 conjugating enzymes, a central domain required for substrate interaction, and an N-terminal E2-like fold (ELF) domain. The ELF domain is found in all FANCL homologues, yet the function of the domain remains unknown. We report here that the ELF domain of FANCL is required to mediate a non-covalent interaction between FANCL and ubiquitin. The interaction involves the canonical Ile44 patch on ubiquitin, and a functionally conserved patch on FANCL. 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.

PubMed ID: 26149689
PMC ID: PMC4543658
Article link: J Biol Chem
Grant support: [+]

Species referenced: Xenopus laevis
Genes referenced: fancd2 fanci fancl ube2l3 ube2t

Article Images: [+] show captions

	FIGURE 1. FANCL binds ubiquitin via the N-terminal E2-like fold. A, pull-down of FANCL species by ubiquitin shows that ubiquitin binding is mediated by the ELF domain. Each experiment is probed with anti-His-ubiquitin and anti-FANCL antibodies, with the input, bait, and beads controls indicated. B, isothermal titration calorimetry curve showing binding of the ELF domain to ubiquitin. Dissociation constant and stoichiometry of the interaction are indicated.
	FIGURE 2. Structural assignment of the Drosophila ELF domain. A, superposition of the ELF domain from Drosophila FANCL in blue (PDB 3K1L) (19) with the E2 Ube2L3 in yellow (PDB 1FBV) (48). E2 protein-protein interaction surfaces indicated, as is the position of the catalytic cysteine of Ube2L3. B, assignment of the Drosophila ELF domain 1H-15N HSQC. The cross-peaks in the 1H-15N HSQC were assigned to residues in the primary sequence of the Drosophila ELF domain.
	FIGURE 3. Reciprocal titrations of FANCL ELF domain and ubiquitin indicate interaction between both proteins. A, 15N-1H HSQC of the 15N labeled ELF domain during titration of wild type ubiquitin. Wild type ELF domain spectra are denoted in black, with 5:1 ELF to ubiquitin in blue and 1:1 in red. The box is a zoom of a portion of the spectra. B, 15N-1H HSQC of 15N-labeled ubiquitin during titration of wild-type ELF. Wild type ubiquitin spectra are in black, with 5:1 ubiquitin to ELF in blue and 1:1 in red. The box is a zoom of a portion of the spectra.
	FIGURE 4. A solvent-exposed patch on the ELF domain interacts with the hydrophobic Ile44 patch of ubiquitin. A, graphical representation of the shifts in cross-peaks in the spectra of the ELF domain upon titration of unlabeled ubiquitin. B, graphical representation of the shifts in cross-peaks in the spectra of ubiquitin upon titration of unlabeled ELF domain. The y axis represents the percentage decrease in cross-peak height for each residue between the wild-type 1H-15N HSQC and the 1H-15N HSQC recorded with 5:1 15N-labeled protein. C, ribbon diagram of the Drosophila ELF domain (in purple) and ubiquitin (in blue) with residues involved in binding highlighted in red. D, surface representations of the Drosophila ELF domain (in purple) and ubiquitin (in blue) with residues involved in binding shown in red.
	FIGURE 5. Mutation of the ELF domain abolishes binding. A, ITC curves showing lack of interaction between ubiquitin and L81R ELF domain. B, ITC curves showing lack of interaction between the ELF domain and I44A ubiquitin. C, 1H-15N HSQC spectra of wild-type DmELF domain (blue) overlaid with 1H-15N HSQC spectra of DmELF-L81R (red). The overlay shows the structure, fold, and stability of both proteins are comparable. The inset shows a Coomassie-stained gel of the proteins used in these experiments.
	FIGURE 6. Ubiquitin binding is conserved in vertebrates. A, structure-based alignment of the ELF domain from various species of FANCL: Drosophila melanogaster (Dm), human (Hs), mouse (Mm), chicken (Gg), Xenopus tropicalis (Xt), and Danio rerio (Dr). Conserved residues are shaded red, conservative substitutions in orange, semi-conservative substitutions in yellow. Residues involved in ubiquitin-binding are boxed, and the Leu-81/Asn-72 residue is marked with an asterisk. Structural elements are included above the sequence. B, pull-down of Xenopus Tropicalis FANCL by ubiquitin shows that ubiquitin binding is conserved. Each experiment is probed with anti-His-ubiquitin and anti-FANCL antibodies, with the input, bait and beads controls indicated.
	FIGURE 7. Ubiquitin binding is not required for E2 recognition. A, pull-down analysis of the interaction between wild type and L81R Drosophila FANCL and human Ube2T or Ube2T-Ub. Both FANCL species bound Ube2T and Ube2T-Ub to the same extent. B, Western blot analysis of Ube2T autoubiquitination in the absence and presence of Drosophila FANCL WT, L81R, and ΔELF species. All variations of E3 were able to successfully stimulate discharge of ubiquitin from Ube2T onto itself. C, in-gel fluorescence analysis of in vitro FANCD2 monoubiquitination (left). Ubiquitin is fluorescently labeled, with no ATP and no E3 controls, showing the modification of FANCD2. The right panel shown 5 independent replicates, with a Coomassie-stained loading control, and quantification of the level of FANCD2 ubiquitination (bottom).
	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.

References [+] :

Alpi, UBE2T, the Fanconi anemia core complex, and FANCD2 are recruited independently to chromatin: a basis for the regulation of FANCD2 monoubiquitination. 2007, Pubmed