FANCD2-FANCI is a clamp stabilized on DNA by monoubiquitination during DNA repair

Vertebrate DNA crosslink repair is a crucial process that excises toxic replication-blocking DNA interstrand crosslinks1. This pathway fails in the inherited human disease Fanconi anemia (FA), resulting in abnormal development, loss of blood production, and marked cancer susceptibility2. Numerous factors involved in crosslink repair have been identified, and mutations in their corresponding genes cause FA3. A biochemical description of how FA gene products might initiate the removal of DNA crosslinks is now emerging. However, structural insight into this vital process has been limited. Here, we use electron cryomicroscopy (cryoEM) to determine the structure of a key DNA crosslink repair factor – FANCD2 heterodimerized with FANCI (D2-I). Recombinant chicken D2-I adopts a conformation that is in agreement with the structure of its murine counter-part4. In contrast, the activated monoubiquitinated form of D2-I (ubD2-I) adopts an alternative closed conformation, creating a channel that encloses double-stranded DNA. Ubiquitin is positioned at the interface of FANCD2 and FANCI, and acts as a molecular pin to trap ubD2-I in the closed conformation, clamped on DNA. The new solvent-exposed interface around the monoubiquitination site is likely a platform for recruitment of other DNA repair factors. We also find that isolated FANCD2 is a dimer, but it adopts a closed conformation that is unable to bind DNA. When incubated with free FANCI, the FANCD2 homodimer exchanges to D2-I, acquiring DNA binding properties. This suggests an autoinhibitory mechanism that prevents FANCD2 activation on DNA until after assembly with FANCI. Together, our cryoEM and biochemical analysis suggests that D2-I is a clamp that is locked onto DNA by ubiquitin, and provides unanticipated new insight into the regulation and the initiation of DNA cross-link repair.

Ubiquitin is positioned at the interface of FANCD2 and FANCI, and acts as a molecular pin to trap ubD2-I in the closed conformation, clamped on DNA. The new solvent-exposed interface around the monoubiquitination site is likely a platform for recruitment of other DNA repair factors. We also find that isolated FANCD2 is a dimer, but it adopts a closed conformation that is unable to bind DNA. When incubated with free FANCI, the FANCD2 homodimer exchanges to D2-I, acquiring DNA binding properties. This suggests an autoinhibitory mechanism that prevents FANCD2 activation on DNA until after assembly with FANCI. Together, our cryoEM and biochemical analysis suggests that D2-

I is a clamp that is locked onto DNA by ubiquitin, and provides unanticipated new insight into the regulation and the initiation of DNA cross-link repair.
A key step in the FA DNA crosslink repair pathway is the monoubiquitination of the FANCD2-FANCI heterodimer [5][6][7][8][9] . Monoubiquitination occurs during DNA replication and is stimulated by the FA core complex -a multi-subunit DNA damage inducible monoubiquitin ligase. Activated ubD2-I localizes to sites of DNA damage and recruits nucleases to remove the lesion 10-17 . Monoubiquitination is tightly controlled, and can be recapitulated in cell free systems in the presence of DNA 18,19 . ubD2-I must be deubiquitinated by USP1/UAF1 for completion of crosslink repair [20][21][22] . A longstanding mystery is why D2-I monoubiquitination and its subsequent reversal should be so fundamental for crosslink repair. Moreover, the mechanistic details of FANCD2 and FANCI interaction with DNA, and how this might ultimately enable DNA crosslink removal remain unclear. Here, we have expressed, purified, and monoubiquitinated Gallus gallus FANCD2 and D2-I complexes, and, using cryoEM and biochemical assays, we delineated key details of how these critical factors interact with DNA.
First, we purified a recombinant D2-I complex after co-expression of both proteins in insect cells, and incubated it with His-tagged ubiquitin, E1, E2 (UBE2T), recombinant FA core complex 23 and DNA (Fig. 1a;Extended Data Fig. 1a). This resulted in specific monoubiquitination of FANCD2 on K563 23 . We enriched for ubD2-I by affinity chromatography using the His-tag on ubiquitin (Fig. 1b). Next, we used cryoEM to investigate the architecture of both  Table 1). The structure of unmodified D2-I agrees with the previously reported crystal structure of the Mus musculus proteins 4 . FANCD2 and FANCI share a striking structural similarity containing a series of ɑhelices arranged into four superhelical ɑ-solenoid structures (S1-S4) and two helical domains (HD1, HD2). They fold into an overall shape akin to two antiparallel saxophones. In this arrangement, the ubiquitination sites (D2K563 and IK525 for the chicken proteins) are buried within the dimerization interface, indicating that remodeling of the complex is very likely required for access by the FA monoubiquitin ligase complex.
In striking contrast, the structure of ubD2-I is markedly different from the unmodified complex. Symmetry was not obvious in 2D class averages (Fig. 1c), but the 3D reconstruction showed that the overall ubD2-I complex retains some pseudo-symmetric features (Fig. 1d). In the monoubiquitinated form, FANCD2 and FANCI are folded towards each other so the Cterminal domains of each monomer (akin to the bells of the saxophone), wrap around density that we assigned to double-stranded DNA (see below). In support of this, multi-body refinement of ubD2-I showed that there is variability in the relative positions of FANCD2 and FANCI (Supplementary Video 1), and this is consistent with previous data 24 .
We then built homology models of FANCD2, FANCI and ubiquitin, and fitted these into the D2-I and ubD2-I maps (Fig. 2a;. Superimposition of the two models showed that the dimer interface acts as a hinge, allowing the C-termini of FANCD2 and FANCI to swing towards each other, rotating by almost 70° . This conformational change in ubD2-I exposes the K563 monoubiquitination site in FANCD2 on the backside of the hinge, where we unambiguously identified density for ubiquitin in the cry-oEM map (Fig. 2b). Ubiquitin is covalently anchored onto FANCD2 (Fig. 1b) but it docks onto the surface of FANCI, and likely stabilizes the complex. In contrast, the FANCI monoubiquitination site (K525) is only partially exposed (Extended Data Fig. 2c), consistent with inefficient in vitro monoubiquitination of FANCI with recombinant or native FA core complex 18,23,25 (Fig. 1b). In cells, FANCI monoubiquitination is stimulated by phosphorylation but is not essential for DNA crosslink repair 9,26 .
The C-terminal solenoids (S4) of FANCD2 and FANCI, as well as additional unmodeled C-terminal regions, mediate contacts at the new interface (Fig. 2c). This interaction likely stabilizes the C-terminal region of FANCD2, which becomes well ordered in ubD2-I map (Fig.   1d). The repositioned C-terminal domains of FANCD2 and FANCI in the monoubiquitinated complex form a new channel between the two protomers. The channel contained density for ~15-20 bp double-stranded DNA. The DNA extended beyond the channel and we could model a total of 33 bp. The DNA was at lower resolution than the rest of the map, likely because of heterogeneity in the DNA position, and inherent flexibility. DNA makes contacts with both proteins, but it appears to be shifted towards FANCI (Fig. 2a). Contacts are likely mediated largely through interactions between the negatively-charged phosphate backbone and positively-charged surface residues 4 . Interestingly, the bound DNA appears to be kinked next to the C-terminal domains within the ubD2-I complex (Fig. 2d).
DNA is required for monoubiquitination of D2-I in vitro 18,19 but the role of DNA in this reaction was unclear. To further investigate this, we performed monoubiquitination assays of D2-I in the presence of a series of double-stranded DNAs of varying lengths. D2-I was monoubiquitinated in the presence of a 19 bp DNA, but not with a 14 bp DNA (Extended Data Fig.  5a). We also analyzed protein-DNA interactions in the monoubiquitination assays using electrophoretic mobility shift assays (EMSAs). D2-I did not bind to a 14 bp DNA, but interacted efficiently with DNA substrates that were 19 bp or longer (Extended Data Fig. 5a). Therefore, these biochemical data show that the minimum DNA length required for ubiquitination (15-19 bp) correlates with the minimum length required for D2-I binding. Moreover, this agrees well with the length of DNA encircled by ubD2-I in the cryoEM structure, and with the distance between a stalled replication fork and a DNA crosslink 6 .
Together, these experiments explain why and how DNA stimulates FANCD2 monoubiquitination: D2-I binds double-stranded DNA, promoting closure of the heterodimer to expose the K563 monoubiquitination site in FANCD2 and allow access to the activated E2 enzyme.
In addition, the structures and biochemical studies suggest that monoubiquitination of FANCD2 on the backside of the hinge acts as a covalent molecular pin, locking the closed heterodimer on DNA and preventing it from rotating back to the open D2-I conformation. In agreement with this, monoubiquitination results in a small, but reproducible, increase in DNA binding affinity (Extended Data Fig. 5b). To further test this hypothesis, we incubated D2-I with FAM-labeled DNA and then tested the ability of a second (Alexa-labeled) DNA to be incorporated into D2-I by displacing the FAM-DNA. We found that in the absence of ubiquitination, the second DNA is readily incorporated into D2-I. In contrast, when FAM-DNA is used to form the ubD2-I complex, a negligible amount of the competing DNA is incorporated (Extended data Fig. 5d).
A major remaining question was why FANCD2 was not monoubiquitinated in vivo or in vitro in the absence of FANCI 13,18,19 . To address this, we purified FANCD2 and FANCI separately. Purified FANCI ran as a monomer on gel filtration chromatography whereas, unexpectedly, FANCD2 ran as a dimer (Fig. 3a). Next, we assessed their DNA binding capacities and found that FANCI bound DNA efficiently but FANCD2 did not interact with DNA at any of the concentrations we tested (Fig. 3b;Extended Data Fig. 5c). Moreover, FANCD2 was not substantially monoubiquitinated by the FA core complex, and this could not be stimulated by DNA (Extended Data Fig. 6a). To understand the molecular basis of FANCD2 dimerization and gain insight into why it did not bind DNA, we determined the structure of the FANCD2 homodimer using cryoEM to ~4 Å resolution ( Fig. 3c; Extended Data Fig. 1). The FANCD2 dimer was symmetric with each protomer containing the same overall domain architecture as in the heterodimeric complex. Surprisingly, the FANCD2 homodimer was in a closed conformation, more similar to ubD2-I than to D2-I. This provides a molecular explanation for the lack of DNA binding by FANCD2: The DNA binding sites were blocked because it was in a closed conformation that presumably does not open to allow entry of a double-stranded helix.
The FANCD2 homodimer was stable over a gel filtration column, indicative of a high affinity interaction. To investigate how the heterodimeric D2-I complex forms, we mixed purified FANCD2 and FANCI and analyzed their oligomerization state. We found that after incubation with FANCD2, the migration position of FANCI on a gel filtration column shifted to the position of a dimer (Fig. 3a). This suggested that monomeric FANCI can displace one of the FANCD2 protomers in the homodimer. To test this directly, we immobilized FANCD2 on Streptavidin resin and incubated it with untagged FANCI. After washing, FANCI was bound to the beads in approximately 1:1 stoichiometric ratio with FANCD2 (Extended Data Fig. 6b). This is consistent with FANCI displacing one of the FANCD2 protomers to form the D2-I complex. Our results suggest a mechanism of autoinhibition wherein FANCD2 cannot be prematurely recruited to DNA prior to assembly with FANCI. This also raises the interesting possibility that the FANCD2-FANCI interaction may be regulated, and complex assembly is stimulated after DNA damage. Alternatively, it may be indicative of FANCI-independent functions of FANCD2. Interestingly, immunoprecipitation of FANCI from cells pulls down only ~20% of FANCD2, suggesting that there may be a substantial pool of FANCD2 not bound to FANCI 9 .
In summary, our structural and biochemical analysis of FANCD2 and FANCI informs how DNA crosslink repair might be initiated (Fig. 4). FANCD2 adopts a closed conformation that does not interact with DNA. Upon incubation with FANCI, the FANCD2 homodimer readily exchanges into a stable D2-I heterodimer, which adopts an open conformation. D2-I binds DNA, and this causes a hinge-like rotation of FANCD2 and FANCI so they encircle double-stranded DNA (Fig. 4). Transition into this closed conformation exposes a region on the back of the FANCD2 hinge, allowing the FA core complex and UBE2T to access the monoubiquitination site. Ubiquitin acts as a covalent molecular pin to lock the ubD2-I DNA clamp into a closed conformation. Finally, the new FANCD2 surface exposed by DNA binding and 6 the ubiquitinated lysine may act as a platform to recruit downstream DNA repair proteins, including the nuclease incision complex that removes the DNA lesion 6,11,12,27 . To release ubD2-I from DNA, the ubiquitin lock must be removed, providing an explanation for why the deubiquitination step is essential to complete repair 20 . This framework, informed by our structures of FANCD2 and FANCI, will be the focus of future work to fully understand the molecular basis of DNA interstrand crosslink repair.  a Homology models for FANCD2 (blue), FANCI (magenta) and ubiquitin (green) were fit into the ubD2-I cryoEM map. The N-and C-termini, solenoids 1-4 (S1-S4) and helical domains (HD1, HD2) 4 of FANCD2 and FANCI are indicated. The monoubiquitinated lysine in FANCD2 and the lysine that can be monoubiquitinated in FANCI are shown in orange. A model of 33 bp double-stranded DNA (yellow) was also placed into the map. b Close-up view of the monoubiquitination site. The ubiquitin moiety attached to FANCD2 makes extensive contacts with FANCI. c Close-up view of the C-terminal domains (S4) of ubD2-I that clamp around DNA. d Double-stranded DNA is kinked within the ubD2-I complex. FANCD2 purifies as a homodimer that is closed, does not bind DNA, and is not monoubiquitinated. Upon incubation with purified (monomeric) FANCI, this exchanges into a FANCD2-FANCI complex (D2-I) with an open conformation. D2-I binds and encircles DNA, converting the complex into a closed conformation, and thereby acting as a DNA clamp. The ubiquitination site on FANCD2 is exposed in the closed conformation, allowing access to the FA core complex and E2 enzyme. Ubiquitin locks the D2-I clamp in a closed conformation so it is not readily released from DNA.

Cloning, expression and purification
cDNAs encoding full length Gallus gallus FANCI and FANCD2 were synthesized (Ge-neArt) and cloned into pACEBac1. The individual genes were amplified by PCR and cloned into pBIG1a vector using a modified version of the biGBac system, as previously described 28,29 . The combined vector was transformed into EMBacY E. coli competent cells for bacmid generation. The purified bacmid was then transfected into Sf9 cells. The virus was passaged twice before a large-scale culture was infected (5 ml of P2 virus into 500 ml of Sf9 cells at 2 million cells/ml). Cell growth and viability were monitored and cells harvested upon growth arrest (typically on day 3 after infection). A similar strategy was used for individual expression of FANCD2 and FANCI except that the FANCD2 gene contained a C-terminal extension with a 3C protease site and double StrepII tag whereas the FANCI gene contained a Cterminal extension with a 3C protease site and 6x His tag.
For D2-I we took advantage of an excellent protein overexpression to devise a tag-free protein purification protocol based on sequential fractionation combining cation exchange and affinity chromatography. Cells were lysed by sonication in lysis buffer (100 mM HEPES pH 7.5, 300 mM NaCl, 1 mM TCEP, 5% glycerol, EDTA-free protease inhibitor, 5 mM benzamidine hydrochloride and 100 U/ml benzonase). Clarified cell lysate was passed through a HiTrap SP HP cation exchange chromatography column (GE Healthcare Life Sciences) to remove impurities. Flow through containing the unbound ID2 complex was diluted to 150 mM NaCl concentration and loaded onto a HiTrap Heparin HP affinity column (GE Healthcare Life Sciences). Using a shallow NaCl gradient, the ID2 complex was eluted around ~ 500 mM NaCl concentration. The complex was run on a Superdex 200 26/60 column (GE Healthcare Life Sciences) in 50 mM HEPES pH 7.5, 150 mM NaCl and 1 mM TCEP. The fractions containing the complex were pooled and concentrated to ~10 mg/ml and flash frozen for storage at -80 °C. Samples from each step were analyzed by SDS-PAGE using 4-12% NuPAGE Bis-Tris gels (ThermoFisher Scientific) to monitor the purification.
His-tagged FANCI was purified by sequential affinity and size exclusion chromatography. Clarified cell lysate produced as for D2-I was loaded onto a HisTrap HP column (GE Healthcare Life Sciences). Using an imidazole gradient, FANCI was eluted around ~200 mM imidazole concentration. Collected fractions containing FANCI were diluted to ~100 mM NaCl concentration and loaded onto a HiTrap Heparin HP affinity column (GE Healthcare Life Sciences). Using a shallow NaCl gradient, FANCI was eluted around ~500 mM NaCl concentration. FANCI was then run on a Superdex 200 26/60 column (GE Healthcare Life Sciences) in 50 mM HEPES pH 7.5, 150 mM NaCl and 1 mM TCEP. The fractions containing FANCI were pooled and concentrated to ~10 mg/ml and flash frozen for storage at -80 °C.
StrepII-tagged FANCD2 was also purified by sequential affinity and size exclusion chromatography. Clarified cell lysate produced as for D2-I and FANCI was incubated with Strep-Tactin Sepharose High Performance resin (GE Healthcare Life Sciences) for 60 min. The loaded resin was mounted on a glass column and washed twice with lysis buffer before elution with 8 mM D-Desthiobiotin. The elution was then diluted to ~100 mM NaCl concentration and loaded onto a HiTrap Heparin HP affinity column (GE Healthcare Life Sciences). Using a shallow NaCl gradient, FANCD2 was eluted around ~500 mM NaCl concentration. FANCD2 was then run on a Superdex 200 26/60 column (GE Healthcare Life Sciences) in 50 mM HEPES pH 7.5, 150 mM NaCl and 1 mM TCEP. The fractions containing FANCD2 were pooled and concentrated to ~10 mg/ml and flash frozen for storage at -80 °C.

Electrophoretic mobility shift assays (EMSAs)
Fluorescently labeled DNA (6FAM labeled on 3ʹ end, purchased from IDT) was prepared by incubating complementary oligonucleotides (Extended Data Table 2) at 95 °C for 5 min and slowly cooling down to room temperature over ~2 h. For EMSAs, a 20 µl reaction containing 20 nM of 6FAM-DNA was incubated with the indicated concentration of protein in the presence of 50 mM HEPES pH 8.0, 150 mM NaCl and 1 mM TCEP. The reactions were incubated for 30 min at 22 °C. After incubation, 5 µl were directly loaded on a native polyacrylamide gel (6% DNA Retardation, Thermo Fisher) and run at 4 °C using 0.5 X TBE buffer for 60 min.
The gel was then visualized using a Typhoon Imaging System (GE Healthcare). Each binding experiment was repeated three times and the intensity of free DNA was calculated using Im-ageJ 30 for the quantification of the mean intensity and standard deviation between the measurements.

Gel filtration assays
A volume of 300 µl of purified D2-I, FANCD2 and FANCI at 1 µM were sequentially run on a Superdex 200 10/300 column (GE Healthcare Life Sciences) equilibrated in 50 mM HEPES pH 7.5, 150 mM NaCl and 1 mM TCEP. To investigate the FANCD2-FANCI exchange we mixed and incubated 0.5 µM FANCD2 with 1 µM FANCI and FANCD2 for 30 min at room temperature in a total volume of 300 µl. The mix was then run on a Superdex 200 10/300 column (GE Healthcare Life Sciences) equilibrated in 50 mM HEPES pH 7.5, 150 mM NaCl and 1 mM TCEP. Fractions were analysed by SDS-PAGE using 4-12% NuPAGE Bis-Tris gels (ThermoFisher Scientific).

Binding assays
We used purified StrepII-tagged FANCD2 and His-tagged FANCI to probe their interaction. In a total volume of 50 µl per reaction, FANCD2:FANCI at molar ratios 1:0, 1:0.5, 1:1, 1:2, 1:5 and 0:5 were mixed in 50 mM HEPES pH 7.5, 150 mM NaCl and 1 mM TCEP and incubated for 15 min at room temperature. Each reaction was applied to 20 µl of StrepTactin Sepharose High Performance resin (GE Healthcare Life Sciences) equilibrated in the same buffer. The loaded resin was then incubated for 30 min at 4 °C. The unbound fraction was removed and the resin further washed twice using 250 µl of the same buffer. The bound fraction was analyzed by SDS-PAGE using 4-12% NuPAGE Bis-Tris gels (ThermoFisher Scientific).

Electron microscopy and image processing
Three microliters of ~1 µM ubD2-I, D2-I or D2 complex were blotted on plasma cleaned UltraAufoil R1.2/1.3 grids 31 (Quantifoil) for 3-4.5 s and plunged into liquid ethane using a Vitrobot Mark IV. The grids were imaged on a Titan Krios using Gatan K3 detector in superresolution mode for ubD2-I and D2 or in counting mode for D2-I. Additional data was collected for ubD2-I at a tilt of 23° to overcome preferred orientation.
All image processing was performed using Relion3.0/3.1 32 unless otherwise stated. The images were drift corrected using MotionCorr2 33 and defocus was estimated using CTFFIND4 34 . Particles were initially picked manually and 2D classified. Selected classes from the 2D classification were used to autopick particles from the full datasets. After 2-3 rounds of 2D classification, classes with different orientations were selected for initial model generation in Relion. The initial model was used as reference for 3D classification into 4 classes. The selected classes from 3D classification were subjected to auto-refinement with solvent flattening using a soft mask. The defocus values were further refined using CTF Refinement in Relion followed by Bayesian polishing. Another round of auto-refinement was performed on these polished particles. All maps were post-processed to correct for modulation transfer function of the detector and sharpened by applying a negative B factor as determined automatically by Relion. A soft mask was applied during post processing to generate FSC curves to yield maps of average resolutions of 3.8 Å for ubD2-I, 4.1 Å for D2-I and 4.0 Å for D2.
To further improve the maps, we used focused classification and refinement of the ubD2-I and D2-I maps by dividing them into FANCI and FANCD2 regions. The density for DNA in ubD2-I was improved by using focused classification without image alignment.
To analyze the conformational heterogeneity in ubD2-I, we applied multi-body refinement using masks around FANCI and FANCD2 protomers. Three major motions were detected using principal component analysis on the optimal orientations of all the bodies for all particle images in the dataset using relion_flex_analyse 35 (Supplementary Video 1).

Modelling
The crystal structure of Mus musculus FANDC2-FANCI (PDB: 3S4W) 4 was used as a template to generate a homology model of Gallus gallus D2-I in I-TASSER 36 . The homology model was initially fitted manually by visual inspection of the map followed by rigid fitting in UCSF Chimera 37 . The side chains were stubbed except for K563 of FANCD2 and the model was iteratively refined in Coot 38,39 and Phenix 40 . The crystal structure of ubiquitin (PDB: 1UBQ) 41 was rigidly fitted into the unassigned density in the ubD2-I map and refined in Coot.
An idealized dsDNA of 33 bp length was placed and refined in the density observed for DNA in ubD2-I using Coot.

Data availability
CryoEM maps generated during this study have been deposited in the Electron Microscopy Data Bank (EMDB) with accession codes EMD-XXXX (D2-I), EMD-YYYY (ubD2-I), and EMD-ZZZZ (D2-D2). Models generated during this study have been deposited in the protein databank (PDB) with accession codes #### (D2-I) and #### (ubD2-I). All other data are available from the authors upon reasonable request.  Sequences from Gallus gallus (gg), Homo sapiens (hs) and Mus musculus (mm) are shown. Under the sequence alignment, the secondary structure of ggFANCD2 is represented in blue: tubes for ɑ-helices and arrows for β-strands. For comparison, the helices have been labelled according to their equivalent nomenclature in the murine structure of unmodified D2-I, and the solenoid (S) and helical domain (HD) numbers are indicated in light blue boxes 4 . Red signs ("FA") indicate mutations characterized in FA patients, purple signs ("P") indicate phosphorylation sites and the monoubiquitination site K563 is marked in green ("U"). Disordered regions are marked with dashed lines.         6 6 0 6 7 0 6 8 0 6 9 0 7 0 0 7 1 0

T E F A V L T L M M R S S S A V A K N R N K G T G G K Q A S
A S C S T L L T D C D 9 0 0 9 1 0 9 2 0 9 3 0 9 4 0 9 5 0

K L F R E L D E V F S L H G L T K I L D T E M H T E
S F 9 6 0 9 7 0 9 8 0 9 9 0 1 0 0 0 1 0 1 0

A W G F L L S A L V L R L K E P L E E L S S Y L N F S P S F Q C
1 1 4 0 1 1 5 0 1 1 6 0 1 1 7 0 1 1 8 0 1 1 9 0 1 2 0 0 1 2 1 0 1 2 2 0 1 2 3 0 1 2 4 0 1 2 5 0   Sequences from Gallus gallus (gg), Homo sapiens (hs) and Mus musculus (mm) are shown. Under the sequence alignment, the secondary structure of ggFANCI is represented in blue: tubes for ɑ-helices and arrows for β-strands. For comparison, the helices have been labelled according to their equivalent nomenclature in the murine structure of unmodified D2-I, and the solenoid (S) and helical domain (HD) numbers are indicated in light blue boxes 4 . Red signs ("FA") indicate mutations characterized in FA patients, purple signs ("P") indicate phosphorylation sites and the monoubiquitination site K525 is marked in green ("U"). Disordered regions are marked with dashed lines.

R E A D G A G A Q D R V H R G
2 5 0 2 6 0 2 7 0 2 8 0 2 9 0 3 0 0          a Monoubiquitination assays were assembled in the presence of linear double-stranded DNA of differing lengths (10-44 bp). DNA binding was analyzed by EMSA (top) and imaging of the fluorescently labeled DNA. Monoubiquitination efficiency was analyzed by Coomassie blue (middle) and Western blotting the His-tagged ubiquitin (bottom). Controls lacking ubiquitin or DNA are indicated. b Monoubiquitination assays of D2-I were assembled without (left) and with (right) ubiquitin, both in the presence of increasing amounts of a 39 bp doublestranded DNA (0 -1000 nM). Assays were analyzed by EMSA (top, imaging for the fluorescently-labeled DNA) or Western blotting His-tagged ubiquitin (bottom). These data are representative of experiments performed twice. c DNA binding of FANCI, FANCD2, D2-I, and FANCI mixed with FANCD2 was analyzed by EMSAs performed with 20 nM 39 bp doublestranded DNA and 0-140 nM protein. Representative gels of experiments independently performed three times (Fig. 3b) and quantitation of mean intensity of free DNA are shown. Error bars represent the standard deviation. Individual data points are shown and means connected by lines for clarity. Fig. 6. FANCD2 forms a homodimer that does not bind DNA.