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

Protein Expression and Purification

All mutants were generated with the QuikChange site-directed mutagenesis kit (Stratagene). Drosophila melanogaster FANCL constructs and DRWD-RING (105–431) were expressed and purified as described previously (19).

The ELF domain (1–105) was cloned into the pET SUMO vector. Luria Broth (LB) growth medium cultures were grown at 37 °C until A₆₀₀ 0.6–0.8. After reducing the temperature to 16 °C, cells were grown to A₆₀₀ 0.8 before inducing with 250 μm isopropyl β-d-1-thiogalactopyranoside (IPTG). Cells were harvested in 500 mm NaCl, 100 mm Tris, pH 8.0, 250 μm Tris(2-carboxyethyl)phosphine (TCEP), sonicated and clarified before affinity purification using Ni-NTA resin (Qiagen) and overnight cleavage with the SUMO protease, Ulp1, in 50 mm NaCl, 100 mm Tris, pH 8.0, 250 μm TCEP. The flow through was loaded onto a Q Sepharose HP in 50 mm NaCl, 100 mm Tris, pH 8.0, 250 μm TCEP and eluted with a gradient including 500 mm NaCl. The purified ELF domain was flash frozen and stored in 50 mm Tris, pH 8.0, 200 mm NaCl, 250 μm TCEP, and 5% (v/v) glycerol at −80 °C.

Human ubiquitin was cloned into pRSF Duet-1 vector with no purification tag. Luria Broth (LB) growth medium cultures were grown at 37 °C until A₆₀₀ 0.5, at which point expression was induced with 500 μm IPTG for 4 h. Cells were harvested and resuspended in PBS buffer before repelleting and flash freezing in liquid nitrogen. The pellets were resuspended in 1 mm EDTA, 25 mm Tris, pH 8.0 before sonication and clarification. The clarified lysate was filtered and loaded onto Q Sepharose HP (GE Healthcare) anion exchange column equilibrated 25 mm Tris, pH 8.0, 1 mm EDTA. The ubiquitin came off in the unbound fraction, before further purification with size exclusion chromatography 250 mm NaCl, 100 mm Tris, pH 8.0 and subsequent storage at −80 °C.

For His-ubiquitin, human ubiquitin was cloned into the pRSF Duet-1 vector with a N-terminal Hexa-His tag. Expression was induced with 0.5 mm IPTG at A₆₀₀ 0.5 followed by batch affinity purification on Ni-NTA (Qiagen) as above. His-ubiquitin was eluted with 300 mm imidazole and further purified by size exclusion chromatography. All interaction experiments are done using monoubiquitin.

Xenopus laevis STREP-FANCD2 cDNA was a kind gift from P. Knipscheer and J. Walter. We expressed and purified the protein as previously described (24). Briefly, Hi5 cells were infected with baculovirus, and the protein was purified on FLAG resin before elution with 3× FLAG peptide and stored with 5% glycerol at −80 °C.

Xenopus tropicalis

FANCL was expressed and purified as previously described (21).

Pull-down Assays

In a total reaction volume of 1 ml containing assay buffer, 500 mm NaCl, 100 mm Tris, pH 8.0, 250 μm TCEP and 10 μm ZnCl₂, Drosophila FANCL, FANCL L81R, ELF, DRWD-RING, Xenopus FANCL, or FANCL N72R (all 250 nm) was added with an excess of His-ubiquitin (1 μm), His-Ube2T (500 nm) or His-Ube2T-Ub (500 nm). Reactions were left to bind for 1 h on ice. 100 μl of Ni-NTA-agarose (Qiagen) was equilibrated in assay buffer and added to the 1 ml reaction and left on a roller at 4 °C for 1 h. Samples were washed with 10 ml of assay buffer and the agarose resuspended in 100 μl of assay buffer. 50 μl of 2× SDS buffer was added before the samples were boiled. 10 μl of the samples was loaded onto SDS page gel and subjected to Western blotting and probed with appropriate antibodies. Anti-ubiquitin antibody was purchased from DAKO. Anti-Drosophila FANCL and anti-human DRWD antibodies were raised from recombinant proteins by Pettingill Technology Ltd. The anti-His antibody was purchased from GE Healthcare.

NMR Spectroscopy and ELF Assignment

The Drosophila ELF domain and ubiquitin were isotopically labeled by overexpression in minimal medium enriched with [¹⁵N]ammonium sulfate and [¹³C]glucose. For NMR samples the ELF domain and ubiquitin were buffer exchanged into 50 mm NaCl, 20 mm sodium phosphate, pH 6.5. Susceptibility matched NMR tubes (Shigemi, US) were used at all stages. Sample volumes of 330 μl were placed in Shigemi BMS-005V tubes and included 10% D₂O v/v. The majority of NMR experiments were carried out at 600 MHz using a VarianUnity INOVA equipped with a 5 mm HCN z-pulse field gradient probe. Chemical shift referencing was based on the position of the water resonance with the exact value being related to the known relationship of the H₂O resonance with temperature. Unless otherwise stated, all NMR experiments were solvent suppressed to reduce the water signal using WATERGATE that was typically obtained using gradient field strengths of 40–50 G cm⁻¹. All NMR datasets were acquired at 25 °C.

All NMR data processing utilized NMRPipe (25) and analyzed using the CCPN analysis package (26). Most resonances were successfully assigned manually without ambiguity. Backbone chemical shift assignments were achieved for the ELF domain using ¹H-¹⁵N HSQC, CBCA(CO)NH, CBCANH, ¹⁵N-NOESY-HSQC, and ¹⁵N-TOCSY-HSQC experiments. Previous amide chemical shift assignments for human ubiquitin were obtained from the VLI Research, Inc. Web site and were used to assign resonances in the ubiquitin ¹H-¹⁵N HSQC.

NMR Titrations

All titration experiments were recorded in 50 mm NaCl, 20 mm sodium phosphate, pH 6.5. The ¹⁵N-labeled domain (wild type ELF or ubiquitin) was at 0.6 mm in the sample, while the binding partner was titrated in at different molar ratios (10:1, 7:5:1, 5:1, 2.5:1, 1:1). All ¹⁵N-¹H HSQC were recorded for 30 min.

Isothermal Titration Calorimetry (ITC)

Kinetic information of the Drosophila ELF domain binding interactions with ubiquitin was established using the ITC200 microcalorimeter (MicroCal, Northampton, MA). Sample buffers were 50 mm Tris, pH 8.0, 100 mm NaCl. Experiments were carried out at 30 °C. The syringe contained 40 μl of 2.5 mm ubiquitin, with 2.5 μl injections every 200 s. The cell contained 205 μl of 230 μm ELF domain. Cell concentrations were adjusted to a 1:1 stoichiometric interaction and Microcal Origin software version 7.0 was used to determine the dissociation constants (K_d). All measurements were repeated at least twice.

DT40 Cell Studies

FANCL-deficient DT40 cells (fancl^−/−) and fancl^−/− complemented with TAP-tagged wild-type FANCL (TAP-FANCL) were described recently (27). Point mutations L7A, D78A, D78R, L79A, and V80A were generated by site-directed muagenesis (QuickChange II XL site-directed mutagenesis kit; Stratagene). DT40 cell transfections, DT40 subfractionation, Superpose 6 gel size chromatography, and FANCD2 immunoblot analyses were described previously (27). To assess TAP-FANCL interaction with ubiquitin, fancl^−/− cells expressing either TAP-FANCL or TAP-FANCL (L7A, D78R, L79A) were lysed in TAP-buffer (50 mm Tris, pH 8.0, 200 mm NaCl, 0.5 mm EDTA, 0.1% Nonidet P-40, and protease inhibitor mixture (Roche Molecular Biochemicals)). TAP-tagged FANCL variants were immunoprecipitated from 2 mg lysates with 50 μl of IgG-coupled Sepharose (GE Healthcare) and washed extensively with Ub-binding buffer (UbBB) (50 mm Tris, pH 7.4, 150 mm NaCl, 0.1% Nonidet P-40, 2 mg/ml BAS). TAP-FANCL Sepharose beads were homogenized in UbBB and incubated with 10 mg/ml HA-tagged wild type or I44A-mutated ubiquitin for 2 h at 4 °C. Beads were washed with wash buffer (50 mm Tris, pH 7.4, 250 mm NaCl, 0.2% Nonidet P-40). Bound ubiquitin was analyzed by immunoblotting with anti-HA (Bethyl Laboratories).

Ube2T Autoubiquitination Assay

100 μl reactions were setup containing 3 μm His-Ube2T, 227 nm E1, 28 μm His-ubiquitin with either 9 μm of E3 (WT Drosophila FANCL, L81R Drosophila FANCL, or Drosophila DRWD-RING) or water in a buffer containing 5 mm ATP, 50 mm Tris, pH 7.5, 100 mm KCl, 0.5 mm DTT, and 2 mm MgCl₂. Subsequently, 10 μl samples were removed from each assay at 0, 15, 20, and 30 min, and arrested with 10 μl LDS buffer (Invitrogen) containing 400 mm β-mercaptoethanol (β-ME). Half of each sample was subjected to Western blot analysis using an anti-His antibody (GE Healthcare).

Charging Ube2T

Charged Ube2T was prepared by adapting the method described in Ref. 28. Human His-Ube2T C24S and tag-free ubiquitin G76C were purified to homogeneity as before, albeit retaining the His-tag on Ube2T. These were then dialyzed against 50 mm sodium borate, pH 8.0, 1 mm TCEP overnight. The proteins were mixed for 15 min on ice with His-Ube2T at 330 μm and ubiquitin at 1 mm. A stock solution of 1,3 dichloroacetone (Sigma) was prepared in dimethylformamide at 20 mm. This was added to the mixed proteins at 0.8 mm and the samples were left rolling at 4 °C for 1 h. The reaction was quenched with 10 mm β-ME for 1 h. The sample was then loaded onto an SD75 16/60 column (GE Healthcare) and purified, and the protein stored at −80 °C with 10% glycerol.

Monoubiquitination of Xenopus FANCD2

Ubiquitination reactions were performed at 30 °C in a 50 mm Tris, pH 7.5, 100 mm KCl, 2 mm MgCl₂, 5% (v/v) glycerol, and 0.5 mm dithiothreitol (DTT) buffer system. Reactions contained 25 nm of recombinant human E1, 0.5 μm of Ube2T, 1 μm of indicates E3 species, 0.5 μm of Xenopus FANCD2, and 2 mm ATP in 20 μl of final reaction volume.

Complete ubiquitination profile was analyzed using fluorescently labeled ubiquitin (Ub⁸⁰⁰). Ubiquitin (residues 2–76) was expressed and purified bearing a GPLCGS overhang at the N terminus. The cysteine residue in the overhang was targeted for site-specific incorporation of a DyLight™ 800 Maleimide (Life Technologies) dye following the manufacturer's protocol. Labeled species was further subject to cation exchange chromatography and stored at −20 °C as single-use aliquots. All ubiquitination reactions contained 2 μm of Ub⁸⁰⁰ and were terminated by boiling with LDS loading buffer. The samples were resolved by SDS-PAGE and analyzed by direct fluorescence monitoring using Li-COR® Odyssey Infrared Imaging System. Integrated intensities of FANCD2 ubiquitination from five independent experiments were obtained using Image Studio™ (Odyssey) imaging software and plotted using GraphPad Prism®.

FANCL Binds Ubiquitin Non-covalently

The ELF domain of FANCL shares significant structural homology with E2-conjugating enzymes (19). E2s form a catalytic intermediate with ubiquitin via a thioester between the catalytic cysteine and the C terminus of ubiquitin (29). The ELF domain does not possess a catalytic cysteine. However, an additional feature of E2s is that they can also interact non-covalently with ubiquitin (30, 31). Therefore we hypothesized that the ELF domain of FANCL might interact with ubiquitin in a similar manner. To test this hypothesis, we performed a pull-down binding assay using 6× His-tagged ubiquitin as bait (Fig. 1A). His-ubiquitin pulls down both full-length Drosophila FANCL and the isolated ELF domain. In contrast, FANCL lacking the ELF domain (ΔELF) is not pulled down. To further characterize the interaction between the ELF domain and ubiquitin, we measured the affinity of binding using isothermal titration calorimetry (ITC), and determined a dissociation constant of 42 ± 12.0 μm (Fig. 1B).

A Surface-exposed Patch on the ELF Domain Interacts with a Hydrophobic Patch on Ubiquitin

We next wanted to understand the molecular determinants of the interaction between FANCL and ubiquitin. E2s bind ubiquitin non-covalently via a backside interaction that involves residues from the loop connecting strands β2 and β3 (30) (Fig. 2A). The dissociation constant between ubiquitin and the ELF domain suggests that crystallization of the complex would prove challenging. Indeed, despite extensive efforts, we were unable to obtain high-resolution diffracting crystals. Therefore, to understand the mode of ubiquitin binding by the ELF domain and whether it is similar to that seen in E2s, we set out to map the interacting surfaces using Nuclear Magnetic Resonance (NMR) spectroscopy. For both structural studies and ITC, milligram quantities of high-quality protein are required. The mammalian and vertebrate homologues of FANCL are not amenable to large scale soluble expression (20, 23); therefore, we used the more soluble invertebrate ELF domain from Drosophila, which shares ∼65% sequence similarity (19% identity) with the human ELF domain (19). First, we determined the solution structure of the ELF domain. Two-dimensional ¹⁵N-¹H HSQC NMR of the ¹⁵N-labeled ELF domain yielded clear and resolved spectra, with excellent chemical shift dispersion, characteristic of a folded globular domain. We unambiguously assigned 76 out of 104 ELF residues using triple-resonance backbone datasets (Fig. 2B). Once we had determined the positions of each residue of the ELF domain in the spectra, we then titrated in increasing amounts of ubiquitin and recorded changes in the two-dimensional ¹⁵N-¹H HSQC. Upon addition of ubiquitin, resonances were broadened to the extent that they were no longer visible, indicating a specific but transient interaction between the proteins (Fig. 3A). We then performed the reciprocal experiments by titrating increasing wild type ELF domain into ¹⁵N-labeled ubiquitin, and identified the binding site on ubiquitin (Fig. 3B). The interaction surface on the ELF domain involves a surface comprising residues Leu-53, His-54, Leu-74, Leu-76, and Leu-81 (Fig. 4, A and B). The interaction surface on ubiquitin is the Leu8-Ile44-Val70 central hydrophobic patch commonly recognized by ubiquitin-binding proteins (Fig. 4, A and B) (32). These results reveal a novel interaction surface on the ELF domain. This surface is not a relic of the E2-like fold, as it does not coincide with the predicted surface upon overlaying the structures (Figs. 2A and 4, C and D). To assess the requirement for residues in the interaction surfaces, we sought to validate our structural insights. We mutated residues involved in the binding, and assayed the resulting proteins for interaction using ITC. The ELF domain point mutant L81R completely abolishes binding (Fig. 5A), as does the ubiquitin mutant I44A (Fig. 5B). To test whether the leucine to arginine mutation on the exposed solvent-accessible surface of the ELF domain disrupts the folding of the domain, we performed two-dimensional ¹⁵N-¹H HSQC NMR of the ¹⁵N-labeled L81R-ELF domain. These experiments yielded clear and resolved spectra, with excellent chemical shift dispersion, characteristic of a folded globular domain, with small changes compared with the wild-type spectra, consistent with a well-folded, stable, mutant protein (Fig. 5C).

Ub Binding Is a Conserved Function of Vertebrate and Invertebrate ELF Domain

The residues in the ELF domain that are important for ubiquitin binding are not well conserved (Fig. 6A). However, ubiquitin-interacting proteins and motifs such as CUE domains, UBAs, UIMs, and MIUs often share very little sequence homology yet retain functional homology (33). We therefore wanted to determine whether the function of ubiquitin binding is conserved in vertebrate FANCL homologs. We and others had difficulties to make wild type full-length human FANCL, therefore we turned to the Xenopus system. Indeed. Xenopus tropicalis FANCL recapitulates ubiquitin binding (Fig. 6B). Mutation of Asn-72, corresponding to Leu-81 in the Drosophila protein, results in almost a complete loss of interaction with ubiquitin, indicating that the same region in FANCL is required for interaction with ubiquitin (Fig. 6B), and that the interaction is conserved between species.

FANCL-Ubiquitin Binding Does Not Enhance the Interaction with Charged Ube2T or Aid Ube2T Discharge

Several E2-RING E3 ligase interactions are enhanced by the presence of the ubiquitin thioester bound on the E2 (34, 35). Therefore another explanation is that FANCL interaction with ubiquitin enhances the recognition of ubiquitin-charged Ube2T (Ube2T∼Ub). To test this hypothesis, we generated a stable Ube2T∼Ub ester and assayed FANCL binding via pull-down. We observed no difference in the levels of FANCL binding in a comparison between ubiquitin-charged Ube2T and uncharged Ube2T (Fig. 7A). Furthermore, there was no difference in Ube2T∼Ub binding when the ubiquitin binding surface of ELF was mutated. These data suggest that ubiquitin binding does not enhance FANCL's interaction with E2.

Although ubiquitin binding does not enhance interaction with Ube2T∼Ub, it may be required for efficient discharge of the ubiquitin thioester. Since Ube2T auto-monoubiquitinates (22), and FANCL has been shown to stimulate this (22), we performed a thioester discharge assay to test whether the ELF-Ubiquitin interaction is important for this. We incubated full-length Drosophila FANCL, FANCL L81R, or ΔELF with human Ube2T, and monitored levels of Ube2T autoubiquitination. Indeed, addition of FANCL, FANCL L81R, or ELF-deleted FANCL (ΔELF), stimulated discharge of ubiquitin to the same extent (Fig. 7B). This suggests that the ELF domain is neither enhancing discharge nor monoubiquitination and is thus not catalytic.

Ubiquitin Binding Is Not Required for Efficient FANCD2 Ubiquitination in Vitro

We next asked whether FANCL's non-covalent interaction with ubiquitin is important for FANCD2 monoubiquitination in vitro. Wild type FANCL supports FANCD2 monoubiquitination in vitro (Fig. 7C). Mutation of the ubiquitin-binding patch in has no effect on the levels of FANCD2 monoubiquitination, further suggesting that the ubiquitin-binding by the ELF domain is not catalytic.

Ubiquitin Binding by FANCL Is Required for Efficient Monoubiquitination of FANCD2 in Vertebrate Cells

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).

FANCL was originally predicted to adopt a WD40-propeller fold in place of the ELF-DRWD domains (12). In a previous study based on this prediction, mutation of the predicted WD40 repeats, including a large part of the ELF domain, was found to disrupt assembly of the FA core complex (16). Since the core complex is required for efficient FANCL-catalyzed FANCD2 monoubiquitination in cells, we next assessed whether the defective FANCI/FANCD2 monoubiquitination in TAP-FANCL (L7A, D78R, L79A) expressing cells was due to disrupted ELF ubiquitin-binding rather than a destabilization of the FA core complex. We first tested TAP-FANCL ubiquitin-binding employing a cell free system. Affinity purified TAP-FANCL interacted with wild type ubiquitin, that was dependent on the ubiquitin Ile-44 patch. Mutated TAP-FANCL (L7A, D78R, L79A) however showed a significantly reduced ubiquitin binding, confirming that amino acids Leu-7, Asp-78, and Leu-79 encompass the ubiquitin interaction surface on chicken FANCL (Fig. 8F).

Next we assayed the formation of the FA core complexes in DT40 FANCL^−/− cells expressing TAP-tagged FANCL or TAP-tagged ELF patch mutant TAP-FANCL (L7A, D78R, L79A). We observed no difference in the pattern of high molecular weight complex formation (Fig. 7A), indicating that ubiquitin binding is not required for the stable incorporation of FANCL into the core complex. Notably, TAP-FANCL (L7A, D78R, L79A) accumulates on damaged chromatin as efficient as wild type TAP-FANCL, further supporting our data that TAP-FANCL complex formation and integrity is independent of ELF ubiquitin binding (Fig. 8G). Taken together, these data suggest that in vertebrate cells, FANCI/FANCD2 monoubiquitination is dependent on the FANCL interaction with ubiquitin, mediated by the ELF domain.