DNA clamp function of the monoubiquitinated Fanconi anaemia ID complex

Abstract

The ID complex, involving the proteins FANCI and FANCD2, is required for the repair of DNA interstrand crosslinks (ICL) and related lesions1. These proteins are mutated in Fanconi anaemia, a disease in which patients are predisposed to cancer. The Fanconi anaemia pathway of ICL repair is activated when a replication fork stalls at an ICL2; this triggers monoubiquitination of the ID complex, in which one ubiquitin molecule is conjugated to each of FANCI and FANCD2. Monoubiquitination of ID is essential for ICL repair by excision, translesion synthesis and homologous recombination; however, its function remains unknown1,3. Here we report a cryo-electron microscopy structure of the monoubiquitinated human ID complex bound to DNA, and reveal that it forms a closed ring that encircles the DNA. By comparison with the structure of the non-ubiquitinated ID complex bound to ICL DNA—which we also report here—we show that monoubiquitination triggers a complete rearrangement of the open, trough-like ID structure through the ubiquitin of one protomer binding to the other protomer in a reciprocal fashion. These structures—together with biochemical data—indicate that the monoubiquitinated ID complex loses its preference for ICL and related branched DNA structures, and becomes a sliding DNA clamp that can coordinate the subsequent repair reactions. Our findings also reveal how monoubiquitination in general can induce an alternative protein structure with a new function.

Main

FANCI and FANCD2 are paralogous proteins and bind to DNA with a preference for branched structures, including Holliday-junction, overhang and replication-fork DNA4,5,6,7. A previous crystal structure of the mouse ID complex showed that it forms an open, trough-like structure with two basic grooves, one on each protomer7. A 7.8 Å crystallographic map of FANCI bound to splayed Y DNA confirmed that its basic groove is the site of double-stranded DNA (dsDNA) binding, and also identified a probable single-stranded DNA (ssDNA)-binding region7. However, it has not been clear how these DNA-binding activities relate to the function of the ID complex in replication and ICL repair. The structure of the mouse ID complex also showed that the monoubiquitination sites are embedded inside the FANCI/FANCD2 interface7, but it did not shed light on the function of monoubiquitination.

To address these questions, we first collected cryo-electron microscopy (cryo-EM) data on the full-length human ID complex bound to an ICL-containing DNA, which was constructed by crosslinking two modified oligonucleotides with a triazole moiety8,9 (Fig. 1a). Although it has not been clear whether the ID complex recognizes ICLs, the triazole ICL DNA mimics two replication forks converging on an ICL—an event that has been shown to trigger ICL repair in a cell-free Xenopus laevis system2. The initial consensus reconstruction with 231,943 particles extended to 3.4 Å, as determined by the gold-standard Fourier shell correlation procedure10,11 (Extended Data Fig. 1). The map showed an overall structure and a FANCI/FANCD2 interface very similar to those of the mouse ID complex7 (Fig. 1a, Extended Data Fig. 1d). Each protomer consists of N-terminal helical repeats that form a long α–α solenoid (henceforth denoted NTD), followed by a helical domain that reverses the direction of the solenoid, and a C-terminal helical repeat domain (CTD) (Fig. 1d, Extended Data Fig. 1g). The consensus reconstruction showed clear density for FANCI and its bound dsDNA, with ssDNA density extending from it. Clear density was observed for the NTD of FANCD2, but poor density was found for the CTD and its bound dsDNA (Extended Data Fig. 2a). Three-dimensional (3D) classification indicated that the CTD of FANCD2 exhibited substantial conformational flexibility, its relative position shifting by as much as 23 Å owing to rotation within the helical domain (Extended Data Fig. 2b, c). FANCI did not exhibit this flexibility, as its NTD contains a helical protrusion that packs with and stabilizes the CTD7 (Extended Data Fig. 1e). Accounting for the flexibility of the CTD of FANCD2 by using multi-body refinement improved the solvent-corrected resolution of the FANCD2 CTD and its associated dsDNA to 3.8 Å, and that of the remainder of the complex to 3.3 Å (Extended Data Figs. 1, 3).

Fig. 1: Human ID complex bound to ICL DNA.
figure1

a, Overall structure of the ID complex bound to DNA. FANCI, cyan; FANCD2, salmon; ICL DNA, yellow. The monoubiquitination sites are shown as sticks and are labelled. The helical axes of the DNA duplexes are shown as grey lines. The inset shows a schematic of the ICL DNA, with triazole-linked deoxycytidine residues in red (not built into the structure). b, View looking down the left side of the horizontal axis of a. c, Cryo-EM density without temperature-factor sharpening. The orientation is similar to that in a. The DNA model is shown in red and its density is shown in yellow. d, Linear representation marking the domain boundaries of FANCI and FANCD2. The darker hues indicate disordered C-terminal segments that become ordered upon ubiquitination, the dashed lines indicate segments that are disordered in both non-ubiquitinated and monoubiquitinated structures.

The improved maps showed continuous dsDNA density extending from the FANCI groove to the FANCD2 groove (Fig. 1c). The DNA is sharply kinked near the centre, which—based on the lengths of the flanking duplexes—is where the ICL would be. There was no density that could correspond to the 5′ overhang ssDNA of the FANCD2-bound duplex. We modelled the overall DNA with an 18-base-pair (bp) duplex and an 8-nucleotide ssDNA bound to FANCI, and a 15-bp duplex on FANCD2, and refined the model with the composite map option of REFMAC512 (Extended Data Table 1). We did not model the ICL and its immediate surroundings owing to the overall high temperature factor and limited resolution of the DNA density. In the refined model, the duplexes bound to FANCI and FANCD2 are at an angle of approximately 33°. Their helical axes are non-collinear, dislocated laterally by about 14 Å (Fig. 1a). This non-collinear arrangement of the two duplexes is largely unaffected by the conformational flexibility of the CTD of FANCD2 (Extended Data Fig. 2d, e, Supplementary Videos 1, 2).

FANCI binds to DNA using an extended basic groove that consists of parts of the NTD, CTD and helical domain. A 4-bp portion of dsDNA distal from the ICL is bound by a semi-circular constriction between the helical domain and the CTD, whereas the rest of the duplex is bound by the NTD, and the ssDNA runs across the last two helical repeats of the CTD (Extended Data Fig. 4a). The DNA-binding activity of FANCD2 differs, in that the helical-domain portion of its semi-circular groove is acidic and is not involved in binding (Extended Data Fig. 4b–e). Rather, FANCD2 binds to DNA using a localized basic patch on its CTD, which is largely non-overlapping with that of FANCI. The density corresponding to the ICL rests against the NTDs of both FANCI and FANCD2 (Extended Data Fig. 4a, b). DNA binding does not cause any conformational changes, as the 3.4 Å cryo-EM structure of the apo-ID complex—also reported here—is essentially indistinguishable from the ID–ICL complex (Extended Data Fig. 1b, f, Extended Data Table 1).

The ID complex can also associate with replication forks in the absence of ICLs13,14,15, and it is implicated in replication-fork recovery after stalling3,16. Therefore, we also collected cryo-EM data for the ID complex with 5′-flap DNA, a reversed fork-like Holliday junction, and a replication fork, as mimics of DNA structures that can arise during replication. All three substrates contained clear density for a FANCI-bound duplex that extended partway to the NTD of FANCD2 (Extended Data Fig. 5). However, there was essentially no density for a duplex bound to the CTD of FANCD2, even in individual 3D classifications. This suggests that engagement of the CTD of FANCD2 requires a translocation of the helical axes of the two duplexes, because although these DNA substrates have a discontinuity in the DNA backbone, they remain stacked17,18.

Together, these data indicate that the principal dsDNA-binding activity of the ID complex resides with FANCI, and that canonical dsDNA is sufficient for FANCI binding. This may account for observations that FANCI alone can accumulate at active replication forks before stalling15. It is also consistent with the observation that the ID complex exhibits only modest specificity for branched DNA structures in biochemical assays5,7 (Extended Data Fig. 6a). We presume that after FANCD2 has engaged in DNA binding at an ICL or at a related DNA structure, this would probably prevent the ID complex from laterally diffusing along the DNA, stabilizing it at the lesion.

We next investigated the function of monoubiquitination by determining the structure of the monoubiquitinated ID (henceforth IDUb). For this, we constructed a stably transfected HEK-293F cell line overexpressing eight subunits of the Fanconi anaemia (FA) core complex, the ubiquitin ligase responsible for ID monoubiquitination (Extended Data Fig. 6b). Using the purified FA core complex with the ubiquitin-conjugating enzyme UBE2T and ubiquitin-activating enzyme UBE1, we ubiquitinated the ID complex in the presence of ICL DNA or various other DNA molecules that have been shown to promote ubiquitination19,20,21 (Extended Data Fig. 6c, d). We purified the reaction products by preparative size-exclusion chromatography and found that IDUb remained bound to DNA, in contrast to non-ubiquitinated ID (Extended Data Fig. 6e, f). We collected cryo-EM data of IDUb bound to four different DNA molecules: ICL DNA, 5′-flap DNA, nicked DNA and dsDNA. The largest dataset was obtained for the nicked DNA complex, and its 3D auto-refinement with 301,058 particles led to a 3.6 Å consensus reconstruction (Extended Data Fig. 7). After focused reconstructions with three masks (3.4 Å, 3.5 Å and 3.5 Å reconstructions), we refined the model of IDUb bound to a 28-bp DNA duplex at 3.5 Å (Fig. 2, Extended Data Table 1). The reconstructions of the 5′-flap DNA, dsDNA and ICL DNA complexes extend to 3.8 Å, 3.8 Å and 4.4 Å, respectively (Extended Data Fig. 8a–c).

Fig. 2: Monoubiquitinated human IDUb complex bound to nicked DNA.
figure2

a, Overall structure of the IDUb–DNA complex. FANCD2Ub, pink; FANCIUb, cyan; UbI, green; UbD2, red; DNA yellow. The N and C termini are labelled (the N terminus of FANCD2 is obscured in this view). FANCI is oriented as in Fig. 1b. b, View looking down the left side of the horizontal axis of a. c, Cryo-EM density without temperature-factor sharpening. The orientation is as shown in b, but parts of FANCD2 are cropped to reveal the internal DNA (shown in red, with density shown in yellow).

Monoubiquitination induces a new mode of association between FANCI and FANCD2, resulting in the conversion of the open-trough structure to a closed ring with the DNA inside (Fig. 2a). Central to the new mode of heterodimerization are the two ubiquitin molecules, whereby the ubiquitin covalently attached to one protomer binds to the other protomer in a reciprocal fashion.

In the non-ubiquitinated ID complex, FANCI and FANCD2 interact along the length of their NTD solenoids in an antiparallel direction, forming an extended interface that buries a total surface area of around 4,950 Å2. The interface is continuous except for two narrow openings, in which the ubiquitination sites—Lys523 of FANCI and Lys561 of FANCD2—are embedded (Fig. 1a, b). After ubiquitination, this interface opens up through a relative rotation of FANCI and FANCD2 by 59° and translation by 15 Å about an axis that runs through the NTD/NTD interface (Fig. 3a, b). None of the intermolecular contacts of the non-ubiquitinated interface is retained (Fig. 3c). Some of these residues are repurposed for interactions with the ubiquitin of the other protomer, with the reciprocal interactions contributing around 3,250 Å2 of buried surface area (Fig. 3c, d). Other residues are repurposed for new intermolecular contacts in a smaller NTD/NTD interface of around 2,800 Å2.

Fig. 3: Conformational changes of ubiquitination.
figure3

a, Non-ubiquitinated ID (FANCI, cyan; FANCD2, salmon; DNA, blue) superimposed on IDUb (FANCIUb, cyan; FANCD2Ub, magenta; DNA, yellow) by aligning FANCI in each structure. UbD2 is shown in red; UbI is obscured in this view. The rotation axis is shown as a thick black stick with the rotation indicated by a circular arrow. Movement of FANCD2 is indicated by a dashed arrow that links equivalent structural elements. The CTD–CTD interaction region is labelled ‘zipper’. The orientation is similar to that in Fig. 2a. b, View looking approximately down the vertical axis of a with the two complexes rendered as semi-transparent surfaces to reveal the DNA inside. c, FANCI (top left, cyan), FANCIUb (bottom left, cyan), FANCD2 (top right, pink) and FANCD2Ub (bottom right, pink) showing residues (thick sticks) for which solvent accessibility is reduced owing to interactions between FANCI and FANCD2 (top pair, blue sticks), between FANCIUb and FANCD2Ub (bottom pair, red sticks), and between each protein and the ubiquitin of the other protomer (bottom pair, green sticks). Yellow spheres mark ubiquitination sites. d, Close-up view of the ID and IDUb complexes superimposed on the FANCD2 ubiquitination site (top) or on the FANCI ubiquitination site (bottom). Non-ubiquitinated ID is shown in transparent grey. e, The CTD zipper of IDUb looking down the vertical axis of b. Residues unstructured in non-ubiquitinated ID are shown in darker shades and are labelled. The dashed cyan line indicates a loop that is unstructured in both ubiquitinated and non-ubiquitinated complexes. f, SDS–PAGE gel of the de-ubiquitination of wild-type IDUb and mutant IDUb(R1285Q) complexes, both at 940 nM, by USP1–UAF1 (400 nM) at the indicated time points, detected by Coomassie staining (top) or anti-ubiquitin immunoblot (bottom). The positions of the substrates and products are marked. This experiment was performed three times.

The relative rotation of the two proteins also juxtaposes their CTD domains, thereby portions of the previously unstructured C-terminal extensions of FANCI and FANCD2 become structured in a zipper-like intermolecular interaction (Fig. 3e). The zipper involves the α48 helix of FANCIUb forming a coiled-coil with α50 of the newly juxtaposed CTD of FANCD2Ub, as well as the interdigitation of the two chains to form an intermolecular β-sheet, capped by a short helix in FANCIUb and a β-hairpin in FANCD2Ub (Fig. 3e). The zipper buries an additional surface area of approximately 4,350 Å2, completing the transformation of the open trough to a closed ring. The importance of ring closing is underscored by an arginine-to-glutamine mutation at residue 1285 (R1285Q) in FANCI, which is associated with Fanconi anaemia19,22. Arg1285, which is on the zipper β-sheet, forms an intermolecular salt bridge with Glu1365 of FANCD2 in a buried environment, whereas it is unstructured in the non-ubiquitinated ID complex (Extended Data Fig. 7f, g). We tested whether the R1285Q mutation of FANCI destabilizes the zipper interface using de-ubiquitination as a surrogate assay, because the lysine–ubiquitin bonds inside the FANCI/FANCD2 interface should be more accessible in a destabilized heterodimer. As shown in Fig. 3f, the de-ubiquitination of the monoubiquitinated ID complex in which FANCI carries an R1285Q mutation (denoted IDUb(R1285Q)) by the cognate de-ubiquitinating enzyme USP1-UAF1 is indeed considerably faster than that of the wild-type IDUb. This mutation also reduces levels of ID ubiquitination in cells14 as well as in vitro (data not shown), which suggests that the transient closure of the zipper—aided by the mobility of the CTD of FANCD2—may be an intermediate in the ubiquitination reaction that enables UBE2T to access the ubiquitination sites.

The conformation of FANCI does not change substantially in IDUb, but FANCD2 undergoes two conformational changes that are important in the remodelling of the complex. The N-terminal part of the NTD rotates by 38° towards FANCI, which is now farther away, to better embrace the ubiquitin conjugated to FANCI (denoted UbI) and also to form a new interface with FANCI itself, while the CTD rotates 20° relative to its NTD to form the zipper interface with the CTD of FANCI (Extended Data Fig. 9a).

FANCI and FANCD2 use similar concave regions of their NTD solenoids (residues 175–377 and 174–348, respectively) to bind to ubiquitin (Fig. 4a). Both interfaces completely cover the ubiquitin hydrophobic patch that consists of Leu8, Ile44 and Val70 (Fig. 4b, c). Ile44, in various combinations with the other two hydrophobic residues, is key to the binding of diverse ubiquitin-binding domains such as UBA, UBZ, UIM and CUE23, the structures of which are unrelated to the ubiquitin-binding helical repeats of FANCI and FANCD2 (Extended Data Fig. 9b–e). The interactions of FANCD2Ub with UbI extend beyond the hydrophobic patch owing to the aforementioned conformational change of the N-terminal portion of FANCD2Ub, which embraces more of the ubiquitin compared with FANCIUb (1,820 Å2 and 1,450 Å2 buried, respectively) (Fig. 4a–c).

Fig. 4: Interactions with ubiquitin and DNA.
figure4

a, Superposition of the FANCD2Ub/UbI and the FANCIUb/UbD2 interfaces by aligning UbD2 (red) with UbI (green). The protomers that each ubiquitin is attached to are not shown for clarity. The residues of the ubiquitin hydrophobic patch (Leu8, Ile44 and Val70) are shown as sticks for both ubiquitin molecules. The colour scheme is the same as that in Fig. 2a. b, Close-up view of the non-covalent interface between FANCD2Ub (pink) and UbI (green) showing the residues within interaction distance as sticks (darker colours). Yellow dotted lines indicate potential hydrogen bonds. c, Close-up view of the reciprocal interface between FANCIUb (cyan) and UbD2 (green). d, Cartoon, coloured as in Fig. 2a, showing side chains of IDUb that are within contact distance of the DNA as sticks (darker hues). Grey sticks show the helical axes for the three relatively straight segments of the DNA. The majority of FANCD2Ub and parts of FANCIUb are clipped above the plane of the figure to make the DNA visible (see Extended Data Fig. 9f for a list of the side chains shown). e, Left, autoradiogram of the DNA-binding competition time course after 8 μM of unlabelled 67-bp dsDNA was added to the IDUb complex (800 nM) pre-assembled on a 95-bp circular nicked DNA or the corresponding linear nicked DNA, both at 400 nM, with 2 nM of each labelled with 32P. 32P-labelled DNA–protein complexes contain multiple IDUb proteins owing to the length of each DNA molecule. Right, quantification of the fraction of DNA bound. For each time point, individual data points are shown by circles and their mean by a black dash; n = 3.

It has been suggested that one function of ID monoubiquitination may be the recruitment of downstream effectors that contain ubiquitin-binding domains1,24. However, the sequestration of the entire ubiquitin hydrophobic patch on both UbI and UbD2 indicates that the ubiquitin is unlikely to have this role with effectors that contain the aforementioned ubiquitin-binding domains. This is consistent with the observation that the UBZ-containing FANCP nuclease scaffold does not require the ubiquitination of FANCD2 for recruitment to ICL sites25. IDUb could still have a recruitment function through other elements, such as the C-terminal EDGE motifs26, that remain unstructured and accessible. We also cannot rule out the possibility that other factors associated with the stalled fork could induce a conformational change in IDUb that exposes the ubiquitin hydrophobic patch, or dissociates the complex into monomers.

Whereas the monoubiquitination of FANCD2 is essential for the FA pathway, FANCI monoubiquitination can be of minor importance in certain settings14,27; this raises the question of whether the singly-monoubiquitinated ID (denoted IDnonUb:Ub, in which only FANCD2 is ubiquitinated) can also assume the closed-ring structure. Indeed, the cryo-EM structure of IDnonUb:Ub, at a resolution of 4.0 Å, has essentially the same structure as IDUb, with only minor shifts localized to the now-empty UbI-binding site of FANCD2 (Extended Data Fig. 8e–g).

The conformational changes in IDUb result in the remodelling of the bound DNA, converting the non-collinear arrangement of the FANCI- and FANCD2-associated DNA duplexes to a continuous but bent duplex (Fig. 4d, Extended Data Fig. 9f). IDUb uses the FANCI groove and the localized CTD patch of FANCD2 to bind to opposite ends of the DNA, as in the ID–ICL DNA complex. Additional DNA contacts occur near the middle of the DNA, where the extension of the α48 helix of FANCI at the CTD–CTD zipper gives rise to a new semi-circular basic groove into which dsDNA binds (Fig. 4d). This is associated with one of the two DNA bends. The second bend occurs as the dsDNA is redirected by the binding site at the CTD of FANCD2. The new position of the CTD of FANCD2 overlaps with and blocks the ssDNA-binding site of FANCI (Fig. 3b).

We have not been able to locate the DNA nick in the map, which looks indistinguishable from the 3.8 Å map of IDUb bound to canonical dsDNA (Extended Data Fig. 8c). Although this could be due to inadequate resolution, the IDUb–5′-flap DNA map also shows no trace of the ssDNA branch (Extended Data Fig. 8b). This suggests either that IDUb binds to these substrates in multiple registers without a preferred location for the nick or flap, or that it is binding only to the dsDNA arms. The latter seems to be the case with the ICL DNA, the duplex arms of which bind to either end of the clamp (Extended Data Fig. 8a). These findings suggest that IDUb has lost the specificity for branched DNA structures that is seen in non-ubiquitinated ID (Extended Data Figs. 6a, 9g).

This raises the possibility that IDUb functions as a sliding DNA clamp. To address this, we first assembled the IDUb complex on either a circular nicked DNA or on the corresponding linear nicked DNA, then added a 20-fold molar excess of unlabelled 67-bp dsDNA and monitored the DNA binding over a time course. At 2 minutes, 73% of the linear DNA complex had dissociated compared with only 4% of the circular DNA complex, 84% of which persisted at 30 min (Fig. 4e). This indicates that the clamp dissociates from linear DNA by sliding off the end and not by opening up, because in the latter case it would dissociate from circular DNA as well.

Our data reveal that the function of ID monoubiquitination is to completely remodel the association between FANCI and FANCD2, thus expanding our understanding of the roles of monoubiquitination. The functional importance of this remodelling is to convert the ID complex into a clamp that can slide away from its initial location at the ICL or related DNA structures. In principle, this would allow downstream nucleases and other factors to act on the ICL, with the IDUb clamp coordinating the repair reactions, serving as a processivity factor, or protecting the DNA.

Methods

No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.

Protein expression and purification

Full-length human FANCI with a non-cleavable C-terminal His6 tag and full-length human FANCD2 with an N-terminal His6 tag followed by a tobacco etch virus (TEV) protease cleavage site were co-expressed in Hi5 insect cells (Invitrogen, not authenticated, not tested for mycoplasma contamination) using baculovirus. Cells were lysed in 50 mM Tris-HCl, 200 mM NaCl, 5% (v/v) glycerol, 0.5 mM TCEP, pH 8.0, and protease inhibitors. After Ni2+ affinity chromatography and overnight cleavage of the FANCD2 His6 tag by TEV protease, the FANCI and FANCD2 proteins were purified by ion-exchange (MonoQ) chromatography, which dissociated the two proteins. The FANCI and FANCD2 proteins were then combined at a 1:1 molar ratio, and concentrated by ultrafiltration to around 20 mg ml−1 in 20 mM Tris-HCl, 150 mM NaCl, 0.5 mM Tris(2-carboxyethyl)phosphine (TCEP), pH 8.0. For the FA core complex, Flag-tagged FANCA, B, C, E, F, G, L, and FAAP100 were cloned into three modified pCDNA3.1 plasmids with different drug-resistance genes, and were used to transfect HEK 293F cells (Invitrogen, not authenticated, not tested for mycoplasma contamination). A stably transfected cell line with the highest expression of all eight subunits was then adapted for growth in suspension. The FA core complex was purified using anti-Flag M2 agarose beads (Sigma), ion-exchange (MonoQ) and gel-filtration chromatography (Superose 6) in 20 mM Bicine-HCl, 150 mM NaCl, 0.1 mM TCEP, pH 8.0.

Preparation of the IDUb complexes

Ubiquitination reactions, carried out in 20 mM Tris-HCl, 150 mM NaCl, 1 mM dithiothreitol (DTT), pH 8.0, contained 40 μM human Flag-tagged ubiquitin (Fisher Scientific, U-120), 0.5 μM human His6-tagged ubiquitin E1 enzyme (Fisher Scientific, E304050), 2.4 μM human His6-UBE2T E2 enzyme (Fisher Scientific, E2-695), 5 mM ATP, 3 μM core complex, 10 μM ID complex and 30 μM 58-bp nicked DNA, or the other DNA substrates. Reactions of 60 μl volume were set up on ice and incubated at 28 °C for 1–2 h. The reaction products were separated by gel-filtration chromatography (Superose 6) in 20 mM Tris-HCl, 150 mM NaCl, 0.2 mM TCEP, pH 8.0, and concentrated by ultrafiltration. Samples were analysed by SDS–PAGE with NuPAGE 3–8% Tris-acetate gels (Invitrogen) and the monoubiquitinated FANCI and FANCD2 were verified using mass spectrometry. To prepare the ID complex, or the IDnonUb:Ub that is monoubiquitinated only on FANCD2, we took advantage of the FA core complex ubiquitinating the ID complex sequentially starting with FANCD2 (data not shown). We ran ubiquitination reactions in the presence of nicked DNA for shorter time periods (10–30 min), and separated the product ID complex that was mostly singly-ubiquitinated on FANCD2 away from the FANCI and FANCD2 substrates by gel filtration chromatography as with the IDUb complex.

Deubiquitination of the IDUb(R1285Q) complex

The R1285Q mutant of human FANCI, denoted FANCI(R1285Q), was generated using QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent); it was cloned with a C-terminal non-cleavable His8 tag into pFastBac vector, and overexpressed in Hi-5 insect cells. The recombinant protein was purified by nickel-affinity, anion-exchange and gel-filtration chromatography and concentrated by ultrafiltration (Amicon) to 85 μM in 20 mM Tris-HCl, 150 mM NaCl, 10% (v/v) glycerol, 1 mM DTT, pH 8.0. FANCI and FANCI(R1285Q) were monoubiquitinated by the FA core complex in a FANCD2-independent reaction (data not shown). The monoubiquitinated FANCI(R1285Q)Ub and the FANCIUb control were purified by anion-exchange chromatography and concentrated to 26 μM and 8 μM, respectively. Monoubiquitinated FANCD2Ub was prepared by running the IDUb complex on an anion-exchange column (MonoQ), which separates it from FANCIUb, and concentrated to 42 μM. The IDUb and IDUb(R1295Q) complexes were then prepared by mixing FANCIUb or FANCI(R1285)Ub with FANCD2Ub at 1 μM, in 20 mM Tris-HCl, 100 mM NaCl, pH 8.0. After incubation on ice for 10 min, 400 nM of the USP1–UAF1 complex (Boston Biochem, E-568) was added. The final concentration of IDUb or IDUb(R1285Q) in the reactions was 940 nM. Reactions (15 μl) were started at 28 °C. At the indicated time points, a 3-μl aliquot was mixed with 9 μl NuPAGE LDS sample buffer (Invitrogen) and heated at 95 °C for 2 min to stop the reaction. Samples were separated by NuPAGE 3–8% Tris-acetate SDS–PAGE (Invitrogen) in duplicate sets for either Coomassie blue staining (6.5 μl loading) or western blotting (4.5 μl loading; anti-ubiquitin antibody from Santa Cruz, sc-271289).

Cryo-EM sample preparation and data collection

For the non-ubiquitinated ID complex bound to the indicated DNA substrates, the concentrated ID complex was combined with a threefold molar excess of DNA. For ID–ICL DNA, the mixture was diluted to 2 mg ml−1 ID (6.5 μM) and 0.8 mg ml−1 ICL DNA (19.5 μM) in 20 mM Tris-HCl, 150 mM NaCl, 0.5 mM TCEP, pH 8.0. For the other DNA substrates, the ID–DNA mixture was diluted to 3 mg ml−1 ID (10 μM) and 27 μM DNA. The samples (3 μl) were applied to glow-discharged UltrAuFoil 300 mesh R1.2/1.3 grids (Quantifoil). Grids were blotted for 1.5 s at 16 °C or 22 °C and around100% humidity and plunge-frozen in liquid ethane using a FEI Vitrobot Mark IV. For all the monoubiquitinated IDUb–DNA complexes, the ubiquitination reactions, performed as described above, were fractionated by gel-filtration chromatography (Superose 6), and the peak corresponding to the IDUb–DNA complex was concentrated by ultrafiltration to around 1.5 mg ml−1 in 20 mM Tris-HCl, 150 mM NaCl, 0.2 mM TCEP, pH 8.0. Grids were prepared as with the non-ubiquitinated complex. All data were collected with a Titan Krios microscope operated at 300 kV and Gatan K2 Summit camera. Most data were collected with a pixel size of 1.089 Å and 10.0 electrons per pixel per second at the Memorial Sloan Kettering Cancer Center (MSKCC) Cryo-EM facility, with additional data collected at the New York Structural Biology Center (NYSBC) Simons Electron Microscopy Center (1.09 Å pixel size), and at the Howard Hughes Medical Institute (HHMI) Cryo-EM facility (1.04 Å pixel size and 8.0 electrons per pixel per second).

Cryo-EM image processing

The super-resolution videos were initially aligned with MOTIONCOR228, and the contrast transfer function (CTF) parameters were estimated with CTFFIND429. All 2D/3D classifications, 3D refinements and other image processing were carried out with RELION-310. For the data from the ID–ICL DNA complex and all the IDUb–DNA complexes, Bayesian beam-induced motion correction, scale and B-factors for radiation-damage weighting, and per particle refinement of CTF parameters were also applied30. All reported map resolutions are from gold-standard refinement procedures with the Fourier shell correlation (FSC) = 0.143 criterion after post-processing by applying a soft mask. For the non-ubiquitinated ID–DNA complexes, the DNA density in the initial 3D reconstructions was weaker than that of the protein, even with low-passed maps, which suggests partial DNA occupancy. We thus used partial signal subtraction outside the FANCI-bound DNA duplex followed by 3D classification without alignment of the density inside the DNA mask, all with RELION-3 (Extended Data Fig. 1b). The fraction of the dataset that contained FANCI-bound DNA ranged from 48% for the ICL DNA complex to 28% for the 5′-flap DNA (Extended Data Figs. 1b, 5a, f, k). The thus-separated particles were then restored and used for the consensus 3D reconstruction of the ID–DNA complexes. For the reconstruction of the apo-ID complex, we used the DNA-free particles from the ID–ICL DNA dataset (Extended Data Fig. 1b, right part of the flow chart). The discord between the electrophoretic mobility shift assay (EMSA)-derived low nanomolar dissociation constant (Kd) values and the partial DNA-occupancy in the cryo-EM maps is probably due, at least in part, to the substantial non-specific DNA-binding activity that the non-ubiquitinated ID complex exhibits in EMSA as described below (see section ‘DNA-binding assays’). Because the non-ubiquitinated ID complex exhibits substantial conformational flexibility in the helical domain and the CTD of FANCD2, we performed multi-body refinement with two soft masks in RELION-3. The larger-body mask (body1 in Extended Data Fig. 1b, c) covered FANCI, the FANCI-bound dsDNA and ssDNA, and residues 45 to 623 of FANCD2, and the smaller body mask (body2) covered FANCD2 residues 624 to 1376 and the FANCD2-associated dsDNA. Multi-body refinement was carried out with signal subtraction outside the masks. The monoubiquitinated IDUb–DNA complexes appeared to have full occupancy of DNA, except for the IDUb–ICL DNA complex that has dsDNA arms shorter than those of the other DNA substrates (Extended Data Fig. 8a). IDUb does not exhibit conformational flexibility in the CTD of FANCD2. The three focused 3D refinements of the IDUb-nicked DNA data improved the resolution only marginally, but there was noticeable improvement in side-chain density and continuity. Of the three partially overlapping masks, one mask (focus1 in Extended Data Fig. 7b, c) covered FANCI residues 288–1297, FANCD2 residues 45–466, UbI, and the FANCI-proximal half of the DNA, corresponding roughly to the left half of Fig. 2b. A second mask (focus2) covered the C-terminal portions of FANCI (residues 598–1298) and FANCD2 (residues 625–1400) and the entire DNA, corresponding roughly to the top half of Fig. 2a. The third mask (focus3) covered FANCI residues 1–376, FANCD2 residues 447–1400, UbD2, and the FANCD2-proximal half of the DNA, and corresponds roughly to the right half of Fig. 2b. For the singly-ubiquitinated IDnonUb:Ub-nicked DNA complex, we removed residual particles containing FANCIUb from the partial ubiquitination reaction using partial signal subtraction outside the UbI softmask, followed by 3D classification without alignment. Subsequent 3D auto-refinement and focused refinements of the particles lacking UbI were performed as with the IDUb complex (Extended Data Fig. 8e, f).

Cryo-EM structure refinement

Model refinement was performed with REFMAC5 modified for cryo-EM12 and with PHENIX31. For the ID–ICL DNA and apo-ID complexes, the two multi-body refinement maps were combined with the composite sfcalc option of REFMAC5 to construct a single set of structure factors to 3.3 Å, the resolution of the larger body1, and refinement was carried out at this resolution. As the resolution of the smaller body2 map is lower (3.8 Å), this results in higher temperature factors for the portion of the model within this map. The model was built on the basis of the structure of the mouse ID complex. Because the DNA has considerably higher temperature factors than the overall protein, it was modelled as dA–dT base pairs. The model was first refined in reciprocal space using REFMAC5, then in real space with PHENIX, and again in reciprocal space including Translation/Libration/Screw (TLS) refinement. REFMAC5 refinement included secondary structure restrains generated by ProSMART12. The structure factors for the IDUb-nicked DNA complex were calculated by combining the three focused 3D reconstructions similarly, except refinement was carried out at 3.48 Å, the highest resolution common to all three maps. The coordinates were assigned to the three focused maps as follows: FANCI residues 327–959, FANCD2 residues 45–454, and UbI to focus1; FANCI residues 960–1297, FANCD2 residues 1150–1400, and the DNA to focus2; FANCI residues 1–326, FANCD2 residues 455–1145, and UbD2 to focus3. Refinement was carried out with REFMAC5 and PHENIX as with the ID–ICL DNA complex. The IDnonUb:Ub-nicked DNA complex was refined similarly.

The refined ID–ICL DNA model lacks the following unstructured regions: 250–259, 401–408, 551–574, 685–695, 935–948, 1111–1125, 1222–1246, 1281–1328 (C terminus) of FANCI, and 1–44, 122–129, 313–337, 589–604, 708–725, 852–915, 947–959, 982–1000, 1043–1050, 1146–1149, 1216–1219, 1377–1451 of FANCD2. As discussed in Fig. 3e, in the refined IDUb-nicked DNA model FANCI residues 1233–1246 (α48 extension) and 1281–1297 (C-terminal residues), and FANCD2 residues 1377–1400, become ordered as part of the zipper interface. The unstructured regions of the non-ubiquitinated ID–ICL DNA complex generally correspond well with the unstructured regions in the crystal structure of the mouse ID complex, or the regions deleted from the mouse FANCD2 construct used in those crystallization experiments based on susceptibility to limited proteolysis7.

DNA-binding assays

EMSA assays were performed using the 40- to 42-bp (or equivalent) DNA substrates shown in Supplementary Table. The non-ubiquitinated ID complex exhibits considerable non-specific DNA binding that seems to correlate with the length, and thus total charge, of the DNA. To try to mitigate that, we used unlabelled 20-bp dsDNA as a non-specific competitor. However, we cannot rule out the possibility that the Kd value of a substrate such as Holliday-junction DNA, which differs considerably from the other substrates in terms of the number of nucleotides, has a larger contribution from non-specific DNA binding. Reactions (15 μl) were assembled by mixing the indicated 32P-labelled DNA substrates (0.5 nM) with the unlabelled 20-bp dsDNA (1.4 μM) and adding the ID complex in 20 mM Tris-HCl, 150 mM NaCl, 5% glycerol, 0.1 mg ml−1 BSA, 0.5 mM TCEP, pH 8.0. They were incubated on ice for 30 min, followed by electrophoresis at 4 °C on 4% (w/v) polyacrymide gels in 0.5× TBE buffer. The dried gels were quantified using a phosphorimager, and the data were fit to a one-site cooperative binding model by minimizing the sum of square of the differences. For the monoubiquitinated IDUb complex, the DNA that was carried over from the ubiquitination reaction and remained bound to the IDUb complex during gel-filtration was removed by anion-exchange chromatography. This also dissociated the FANCIUb and FANCD2Ub proteins, which were then concentrated separately by ultrafiltration. Binding reactions were assembled by first mixing FANCIUb with the indicated 32P-labelled DNA substrate (0.5 nM) and then adding FANCD2Ub at a 1:1 molar ratio to FANCIUb. The subsequent steps were performed as with the non-ubiquitinated ID complex. The IDUb reactions did not contain the unlabelled dsDNA competitor used with the non-ubiquitinated ID complex. For the competition experiments in Fig. 4f, DNA-binding reactions were assembled by first adding monomeric FANCD2Ub (800 nM) to the indicated 5′ 32P-labelled DNA (400 nM, with only 2 nM labelled), then adding FANCIUb (800 nM) in the same buffer as above. Reactions were incubated on ice for 30 min before adding a 20-fold molar excess (to the substrate DNA) of unlabelled 67-bp dsDNA competitor (8 μM) to start the time course, followed by electrophoresis at 4 °C as above.

DNA substrates

All of the DNA substrates were prepared by annealing the oligonucleotides listed in the Supplementary Table. The 95-nt circular oligonucleotide was synthesized by Bio-Synthesis Inc. Its sequence is based on a 91-nt case study on the Bio-Synthesis website, except for the addition of 4 nts to create a restriction enzyme site. The double-stranded circular and its corresponding linear DNA molecules were prepared by annealing complementary oligonucleotides. The ICL DNA was prepared as described9. In brief, we used the Cu(i)-catalysed azide–alkyne cycloaddition between an N4-(3-azidopropyl) modified cytosine on a dCdG step of one DNA strand, and an N4-propargyl modified cytosine on the complementary strand, crosslinking the N4 positions of the two cytosines with a triazole moiety. The A3 and A4 oligonucleotides were synthesized by Sigma incorporating N4-(3-azidopropyl) deoxycytidine and N4-propargyl deoxycytidine, respectively, at the positions indicated by the bold ‘C’. For the A3 oligonucleotide, the synthesis involved first adding an N4-chloropropyl deoxycytidine phosphoramidite to avoid an azide–phosphoramidite side reaction, with the chloropropyl group subsequently converted to azidopropyl on the beads. The oligonucleotides were purified by HPLC by Sigma. For the crosslinking, the two oligonucleotides, each at a concentration of 0.2 mM, were incubated with 2 mM CuSO4, 10 mM sodium ascorbate and 1 mM Tris(3-hydroxypropyltriazolylmethyl)amine in 10 mM HEPES-Na, 50 mM NaCl, pH 7.5, for 30 min at room temperature. The reaction products were separated by denaturing PAGE (12% polyacrylamide, 8 M urea), and the crosslinked product was isolated from the gel slice by electroelution (Whatman Elutrap). The ICL was confirmed by liquid chromatography coupled with electrospray ionization mass spectrometry, performed by Novatia.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.

Data availability

The ID–ICL DNA, IDUb–DNA, IDnonUb:Ub–DNA and apoID coordinates, corresponding cryo-EM maps, including the focused reconstructions and the composite map used in refinement, have been deposited with the Protein Data Bank (PDB) and the Electron Microscopy Data Bank (EMDB) under accession codes PDB-6VAA and EMDB-21134, PDB-6VAE and EMDB-21138, PDB-6VAF and EMDB-21139, and PDB-6VAD and EMDB-21137, respectively.

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Acknowledgements

We thank the staff of the MSKCC Cryo-EM facility, the NYSBC Simons Electron Microscopy Center, and the HHMI Cryo-EM facility for help with data collection. This work was supported by the HHMI and National Institutes of Health grant CA008748.

Author information

Affiliations

Authors

Contributions

R.W. and S.W. collected and analysed the cryo-EM data; R.W. performed protein purification, ubiquitination reactions and DNA-binding competition experiments; S.W. prepared the FA core complex and performed the de-ubiquitination assays; A.D. collected cryo-EM data and performed DNA-binding EMSA assays and protein purification; C.P. performed DNA-binding EMSA assays and protein purification; and N.P.P. analysed the data and wrote the manuscript.

Corresponding author

Correspondence to Nikola P. Pavletich.

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The authors declare no competing interests.

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Peer review information Nature thanks xxx for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Cryo-EM reconstructions of the non-ubiquitinated ID–ICL DNA and apoID complexes.

a, Micrograph of non-ubiquitinated ID–ICL DNA particles (4,038 Å by 4,178 Å magnified micrograph dimensions). The presence of excess DNA at 0.8 mg ml−1 obscures the protein particles. The particles were collected in five datasets. b, Flow chart of single particle cryo-EM data processing for the ID–ICL DNA and apo-ID complexes. Consensus (top) and focused maps from the RELION3 multi-body refinement, temperature-factor sharpened and masked, are coloured by local resolution estimated with the RELION3 postprocess program. The resolution range is mapped to the colours in the inset next to each map. Orientation is similar to that in Fig. 1a. The particle has dimensions of 164 Å, 116 Å, 96 Å. For additional details, see Methods. c, The top graph shows gold-standard FSC plots between two independently refined half-maps for the ID–ICL DNA consensus reconstruction (blue curve), for the RELION3 multi-body refinement of the larger body1 consisting of FANCI, FANCI-bound dsDNA and ssDNA, and FANCD2 residues 43–623 (red curve), and for the smaller body2 consisting of FANCD2 residues 624–1376 and the FANCD2-associated dsDNA (green curve). The FSC curve for the final model versus the composite map combining the cryo-EM maps of the two bodies is shown in black. The dashed line marks the FSC cutoff of 0.143. The bottom graph shows the gold-standard FSC plots between two independently refined half-maps for the consensus reconstruction of the apo-ID complex (blue curve), of the RELION3 multi-body refinement of the two bodies (red and green curves), and of the model refined with REFMAC5 (black). d, Top left, superposition of the structure of human FANCI on the structure of mouse FANCI from the crystal structure of the mouse ID complex28, with a root-mean-square deviation (r.m.s.d.) of 1.8 Å for 926 out of 1,168 common Cα atoms (coloured blue and purple for human and mouse, respectively). The majority of the residues that do not superimpose are in the N-terminal 183-amino-acid segment that packs with the FANCD2 helical domain that is mobile in the cryo-EM structure. Other differences include FANCI residues 551–574, which are disordered in human FANCI but the corresponding mouse FANCI segment (marked by a dotted oval and labelled ‘P-site loop (mouse)’) is well ordered at the FANCI/FANCD2 interface, and affects the conformations of flanking helical repeats. The FANCD2 region, with which this segment packs in the mouse ID complex, is shifted by approximately 5 Å away from FANCI in the human ID complex. This segment also contains the phosphorylation sites of ATR kinase7,27. Ubiquitination sites, N- and C- termini and individual domains are marked. Top right, superposition of the structure of human FANCD2 on the structure of mouse FANCD2 with an r.m.s.d. of 2.0 Å for 501 out of 1,131 common Cα atoms (coloured pink and green for human and mouse, respectively). The low level of overall structural overlap is due to the flexibility of the CTD and helical domain of FANCD2 in the cryo-EM structure compared to the mouse ID crystal structure, in which the CTD of FANCD2 is involved in crystal packing contacts that limit its mobility. Another difference involves the first approximately 200 residues that cap the NTD solenoid of FANCD2. This segment is displaced by 5 Å in the mouse structure owing to the ordering of the aforementioned FANCI phosphorylation-site loop at the interface. Bottom, the superposition of the human ID complex on the crystal structure of the mouse ID complex with an r.m.s.d. of 2.2 Å for 1,302 out of 2,312 common Cα atoms. The superimposed segments are primarily the NTD solenoids of FANCI and FANCD2, including their ubiquitination sites but excluding their N termini as discussed above, and most of the CTD domain of FANCI. The dashed oval marks the helical protrusion (NTD–CTD bridge) from the NTD of FANCI, which packs with its CTD and rigidifies the structure. The lack of an NTD–CTD bridge in FANCD2 is associated with the increased mobility of its CTD relative to the NTD. e, Expanded view of the FANCI NTD–CTD bridge of the human and mouse structures, coloured red and yellow, respectively. f, DNA binding does not cause any substantial conformational differences, except for in the CTD of FANCD2, which exhibits increased mobility in apo-ID complex. The human ID–ICL complex (FANCI, cyan; FANCD2, pink) can be superimposed on the human apo-ID complex (FANCI, purple; FANCD2, green) in its entirety with a Cα r.m.s.d. of 0.9 Å (2,291 common Cα atoms). g, The human ID–ICL DNA complex coloured according to the domains of the individual proteins as indicated, in the same orientation as Fig. 1a.

Extended Data Fig. 2 Conformational flexibility of the CTD domain of FANCD2 and its associated DNA.

a, The CTD of FANCD2 and its associated DNA are evident in the consensus reconstruction before temperature-factor sharpening. Because the DNA has higher temperature factors than the protein (Extended Data Table 1), the temperature-factor calculated from the overall map degrades the continuity of the DNA density. The cartoon representation of the refined model is coloured as follows: FANCI, cyan; FANCD2, pink; DNA, gold. The schematic of the ICL DNA is shown to the right of the map, with the deoxycytidine bases that are crosslinked by a triazole coloured red. The 20-nt ssDNA arms consist of (dT)20 to minimize secondary structure. b, 3D classification of the particles showing the conformational flexibility of FANCD2. The 3D classes are arranged starting with the most compact conformation in which the CTD of FANCD2 is closer to its NTD. Also shown is the refined consensus model rigid-body-fitted into each class and coloured as in a. The conformational flexibility starts within the helical domain (starting around residue 645). c, The five 3D classes are superimposed by aligning the FANCI portion of each map (top), or of each PDB structure (bottom) coloured according to their map in b. d, Cryo-EM reconstructions using particles from the top component of the principal component analysis (PCA) of the multi-body refinement angles, separated into five bins. This component accounts for 21.5% of the variance in the relative orientation of the CTD of FANCD2 (Supplementary Video 1). Left, five ID–ICL DNA models refined in real-space with PHENIX (overall solvent-corrected resolution ranging from 3.7 to 3.9 Å) against maps reconstructed with particles derived from five bins of eigenvalues for the top eigenvector. This PCA component corresponds to a rotation of up to 16° (curved arrow) about an axis running through the helical domain that is roughly perpendicular to the plane of the figure (grey stick). The helical axes of the individual duplexes are shown as black sticks. Right, the corresponding maps, without temperature-factor sharpening, starting with the conformation (pink map) in which the CTD of FANCD2 is closest to the CTD of FANCI. e, The second component from the PCA accounts for 17% of the variance in the relative orientation of the CTD of FANCD2 (Supplementary Video 2). It corresponds to a rotation of up to 10° (curved arrow) about an axis (grey stick) that is roughly parallel to the plane of the figure. Left, the refined models; right, the maps as in d.

Extended Data Fig. 3 Cryo-EM density from post-processed maps of non-ubiquitinated ID bound to ICL DNA.

a, b, Stereo view of the 3.3 Å cryo-EM density from the post-processed reconstruction using multi-body refinement. Map shows the vicinity of the FANCI ubiquitination site (Lys523 marked) with portions of FANCI residues 475–593 (cyan) and FANCD2 residues 174–287 (pink) shown in stick representation (a) and the FANCD2 ubiquitination site (Lys561 marked) with portions of FANCD2 residues 482–578 and FANCI residues 123–223 (b). Oxygen and nitrogen atoms are coloured half-bonded red and blue, respectively, for both proteins. c, Stereo view of the map from a focusing on the ssDNA (5′ and 3′ ends marked) at the CTD of FANCI, as well as a portion of the FANCI-bound dsDNA. The DNA is in stick representation coloured half-bonded yellow, red and blue for carbon, oxygen and nitrogen atoms, respectively. The map is shown at a low contour level because the ssDNA has high temperature factors, and its density is broken up owing to the temperature-factor applied in post-processing being calculated from the entire map. ssDNA density before post-processing can be seen in the panel of maps in Extended Data Fig. 2d, e. d, e, Mono view of the 3.3 Å cryo-EM density depicted as a semi-transparent surface at the hydrophobic core of the NTD of FANCI, showing the residues 202, 214, 217, 236–237, 271–272 and 300 (d), and the NTD of FANCD2, showing the residues 347, 364, 368, 380 and 383–386 (e).

Extended Data Fig. 4 DNA binding by the non-ubiquitinated ID complex.

a, DNA binds to an extended basic surface of FANCI. Cartoon representation showing FANCI side chains within potential contact distance of the DNA (top), and molecular surface coloured according to the electrostatic potential calculated with PyMOL (bottom, coloured −5 to +5 kT blue to red). The proteins are coloured according to their domains, in purple, cyan and grey for the NTD, CTD and helical domain of FANCI, respectively, and brown, pink and grey for the NTD, CTD and helical domain of FANCD2, respectively. The end of the dsDNA binds to a semi-circular groove that consists of helices α33b, α36b, α37, α40 and α42 (secondary structure elements numbered as in the structure of the mouse ID complex28, with insertions denoted by letters after the helix number). This is analogous to the 7.8 Å crystallographic map of mouse FANCI bound to Y DNA, with the N termini of helices and inter-helix loops providing multiple basic residues. The ICL-proximal portion of the duplex, which is absent from the shorter DNA used in the formation of mouse FANCI crystals, is positioned against basic residues emanating from helices α15, α17 and α19b. The ssDNA rests against the sides of the α48 and α49 helices. The overall DNA density is of lower resolution than the surrounding protein, and in the refined model the DNA has high temperature factors suggesting that it is considerably more mobile than the surrounding protein elements. Side chains are shown for Arg287 on α15; Arg321, Lys336 and Lys339 on α17; Lys396 and Lys397 on α19b; Lys791, Lys793, Thr794 and Lys795 on α33b; Lys897, Lys898 and Lys902 on α36b; Lys980 on α40; Lys1026 on α42; Arg1178 on α46; and Lys1262, His1266, Lys1269 and Lys1270 on α48. b, FANCD2–DNA contacts are localized to the last four helical repeats of the CTD and a patch of basic residues on the NTD. Top, the residues within contact distance of the DNA, coloured as in a. The CTD residues involve the N-terminal portions of the inner helices of the helical repeats: Lys1172, Lys1174, Ser1175, Ser1178, Asn1179 and His1183 on α44; Arg1128, His1229 and Arg1236 on α46; Ser1287, His1288, His1292, Lys1296 and Tyr1297 on α48; and Thr1351, Arg1352, Gln1355 and His1356 on α50. NTD residues on α50 are Arg401, Arg404, Asn405 and Arg408. Bottom, the corresponding molecular surface coloured according to the electrostatic potential calculated with PyMOL (bottom, coloured −5 to +5 kT blue to red). Note the absence of a basic patch at the helical-domain portion of the semi-circular groove (lower left) compared to that of FANCI in a. c, Expanded view of the FANCD2 Arg1352 side chain in the DNA minor groove, and the residues that are in the vicinity of the flanking phosphodiester backbone. d, Expanded view of c, showing the 3.8 Å cryo-EM density centred on Arg1352, shown in stick representation. Only a subset of the side chains shown in c are visible in this view (His1288, His1292, Lys1236, Lys1296, Gln1355 and His1356). e, Superposition of the DNA-binding region of the CTD of FANCD2 (pink) on the corresponding region of FANCI (cyan) showing the different orientations of the FANCI dsDNA (blue) and FANCD2 dsDNA (magenta) in the semi-circular grooves of the respective proteins. Residues 905–1269 of FANCD2 were aligned on residues 1058–1376 of FANCI with a 2.2 Å r.m.s.d. in the positions of 185 Cα atoms.

Extended Data Fig. 5 Cryo-EM reconstructions of the non-ubiquitinated ID complex bound to 5′-flap DNA, Holliday junction DNA, and replication fork DNA.

a, Flow chart of single particle cryo-EM data processing. The consensus reconstruction map, temperature-factor sharpened and masked, is coloured by local resolution estimated with the RELION3 postprocess program. The graph on the right shows the gold-standard FSC plot between two independently refined half-maps for the consensus reconstruction with 87,802 particles. The dashed line marks the FSC cutoff of 0.143 that the FSC curve intersects at 4.0 Å. b, Cryo-EM map from the consensus reconstruction without temperature-factor sharpening. Also shown are cartoon representations of the FANCI (cyan), FANCD2 (pink) and FANCI-bound dsDNA (gold) from the ID–ICL DNA complex rigid-body-fitted as multiple domains into the map. Schematic of the 5′-flap DNA is shown to the right of the map. c, 3D classification of the particles showing the conformational flexibility of FANCD2. Maps shown are after the particles from each 3D class were further refined in RELION to 4.7, 4.3, 4.3 and 4.9 Å, respectively. The maps are calculated without temperature-factor sharpening to make the DNA easier to see. The ID complex and dsDNA, rigid-body-fitted into each map are also shown. d, The five 3D classes are superimposed by aligning the FANCI portion of each map (left), or of each PDB structure (right) coloured as in c. The lack of FANCD2-bound dsDNA is indicated by the label ‘~no FANCD2 DNA’. The density of the CTD of FANCD2 is considerably weaker and flatter than the similarly calculated maps of the of the ID–ICL DNA complex. The curved arrow indicates the motion suggested by the 3D classification. In the most compact class (blue map), there is density extending from the CTD of FANCD2 to the CTD of FANCI. Although we could not improve this density owing to the limited number of particles, it suggests that the flexibility of the CTD of FANCD2 may be important for the closing of the structure upon ubiquitination. e, Expanded view of the best 3D class (pink in c) after 3D refinement of the particles before post processing. Orientation is similar to that in Fig. 1c. Neither this map nor those of the other 3D classes have any evidence of a localized fork junction or of the 5′-ssDNA flap, suggesting that the 5′-flap DNA binds to FANCI in multiple registers with no specificity for the junction. f, Flow chart of cryo-EM data processing for the ID complex bound to Holliday junction DNA. The consensus reconstruction map, temperature-factor sharpened and masked, is coloured by local resolution estimated with the RELION3 postprocess program. The graph on the right shows the gold-standard FSC plot between two independently refined half-maps for the consensus reconstruction with 76,690 particles, with an FSC value of 0.143 (dashed line) at 4.1 Å. g, Cryo-EM map from the consensus reconstruction before post processing. Also shown are cartoon representations of the FANCI (cyan), FANCD2 (pink) and FANCI-bound dsDNA (gold) from the ID–ICL DNA complex rigid-body-fitted into the map. A schematic of the Holliday junction DNA used is shown to the right of the map. h, 3D classification of the particles showing the conformational flexibility of FANCD2. Maps shown are after the particles from each 3D class were further refined in RELION to 6.9, 4.7, 6.6, 4.7 and 6.5 Å, respectively. The maps are prepared without temperature-factor sharpening. The ID complex and dsDNA, rigid-body-fitted into each map are also shown. i, The five 3D classes are superimposed by aligning the FANCI portion of each map (left), or of each PDB structure (right) coloured as in h. j, Expanded view of the best 3D class (green in h) after 3D refinement of the particles before post processing. Unlike the map with the 5′-flap DNA, this map shows some bifurcation at one end of the duplex, which is suggestive of the presence of the Holliday junction at a preferred location. This may be due to its shorter duplexes of 20 bp being just long enough to fill the FANCI groove. k, Cryo-EM data processing flow chart of the ID complex bound to replication fork DNA. The consensus reconstruction map, temperature-factor sharpened and masked, is coloured by local resolution estimated with the RELION3 post-process program. The graph on the right shows the gold-standard FSC plot between two independently refined half-maps for the consensus reconstruction with 111,664 particles, with an FSC value of 0.143 (dashed line) at 3.9 Å. l, Cryo-EM map from the consensus reconstruction before post processing. Also shown are cartoon representations of the FANCI (cyan), FANCD2 (pink) and FANCI-bound dsDNA (gold) from the ID–ICL DNA complex rigid-body-fitted into the map. Schematic of the replication fork DNA used is shown to the right of the map. m, 3D classification of the particles showing the conformational flexibility of FANCD2. Maps shown are after the particles from each 3D class were further refined in RELION to 4.8, 4.7, 4.6 and 4.4 Å, respectively. The maps are prepared without temperature-factor sharpening. The ID complex and dsDNA, rigid-body-fitted into each map, are also shown. n, The five 3D classes are superimposed by aligning the FANCI portion of each map (left), or of each PDB structure (right), coloured as in m. o, Expanded view of the best 3D class (orange in m) after 3D refinement of the particles before post-processing.

Extended Data Fig. 6 Biochemical characterization of ID–DNA binding and reconstitution of ID monoubiquitination.

a, EMSA of the ID complex binding to the indicated 32P-labelled DNA substrates (0.5 nM) in the presence of 1.4 μM unlabelled, 20 bp dsDNA as non-specific competitor. The plots with a logarithmic x axis show the fraction bound in three repetitions of each experiment (blue, green and orange markers), and their mean value (black dash). Each binding isotherm fits a Hill slope model considerably better than a non-competitive binding model, even after excluding the highest protein concentration reactions in which multiple shifted bands are apparent, and also in the absence of non-specific competitor DNA (not shown). A binding curve (black line) simulated with the indicated Kd and Hill coefficient (ηH) values is shown on each plot. b, SDS–PAGE gel of the purified FA core complex. m.w., molecular weight markers with their mass labelled; core: FA core complex with the constituent proteins labelled. The core preparation was performed at least three times, and was performed an additional time with a different isolate of a stably transfected cell line with very similar results. c, SDS–PAGE of the ubiquitination reaction of the ID complex in the presence of a 58-bp nicked-DNA molecule and of three peaks from the fractionation of the reaction products on a Superose 6 gel-filtration column shown in d. The gel and gel-filtration run of d are typical of the preparative reaction and purification performed at least three times with nicked DNA and once each for the DNA substrates of Extended Data Fig. 8a–c with similar results. d, Gel-filtration chromatography of the ubiquitination reaction products (blue plot) and of the DNA-only control (orange plot). The fraction marked 1 contains the core complex, fraction 2 the complex of uniquitinated FANCI and ubiquitinated FANCD2, and fraction 3 contains monomeric non-ubiquitinated FANCI and FANCD2, as well as the overlapping peak of excess DNA. e, Comparison of the gel-filtration chromatography profiles of the ID ubiquitination reaction product (blue plot) and of non-ubiquitinated ID (red plot), both at 8 μM, in the presence of 16 μM ICL-DNA and 100 mM NaCl. f, 4% TBE gels of the gel-filtration fractions marked with an arrow in e stained for protein with Coomassie blue and for DNA with SYBR Gold. Compared with ubiquitinated ID, only a residual amount of non-ubiquitinated ID remains bound to DNA, and its DNA and ID–DNA bands are faint. Because gel-filtration chromatography is more sensitive to the off rate of a complex than is the EMSA assay, this suggests that the IDUb complex has a slower off rate, which is consistent with its closed-ring structure compared to the open-trough structure before ubiquitination. Complexes of IDUb with nicked DNA and with the other DNA substrates of Extended Data Fig. 8 behave similarly to the IDUb–ICL complex under these conditions (not shown). The chromatography of e and SDS–PAGE of f were repeated twice with similar results.

Extended Data Fig. 7 Cryo-EM reconstruction of the IDUb complex bound to nicked DNA.

a, Micrograph of IDUb-nicked DNA particles. The particles were collected in three datasets. b, Flow chart of single particle cryo-EM data processing. Consensus (top) and focused maps from RELION3, temperature-factor sharpened and masked, are coloured by local resolution estimated with the RELION3 postprocess program. The focused maps below roughly correspond to the left, top, and right portions of the consensus map. The maps are all oriented as the structure on the right (coloured as in Fig. 2a). The resolution range is mapped to the colours in the inset next to each map. The particle has dimensions of 155 Å, 115 Å, 101 Å. For additional details, see Methods. c, Graph shows gold-standard FSC plots between two independently refined half-maps for the consensus reconstruction of the ID–ICL DNA (blue curve) and the three focused maps. The FSC curve for the final model versus the composite map combining the focused maps in REFMAC5 is shown in black. The dashed line marks the FSC cutoff of 0.143. d, Stereo view of the 3.5 Å cryo-EM density of the isopeptide bond between the ubiquitin Gly76 C atom and the Nζ atom of Lys523 of FANCI from the post-processed reconstruction of the focused refinement. UbI, green; FANCI, cyan. Oxygen and nitrogen atoms are coloured half-bonded red and blue, respectively, for both proteins. Also shown is FANCD2 Trp182 (pink) that packs with Lys523. e, Stereo view of the 3.5 Å cryo-EM density of the isopeptide bond between the ubiquitin Gly76 C atom and the Nζ atom of Lys561 of FANCD2 from the post-processed reconstruction. UbD2, dark red; FANCD2 pink. f, Stereo view of the 3.4 Å cryo-EM density of the zipper β-sheet of the IDUb complex. FANCI, cyan; FANCD2 pink. The arrow points to Arg1285 of FANCI, which is mutated to glutamine in Fanconi anaemia. Select hydrogen bonds (made by the β-sheet and by Arg1285) are shown as green dotted lines. g, Mono view of the density in f, rendered semi-transparent, focusing on the vicinity of Arg1285 of FANCI. Arg1285 and Glu1365 of FANCD2, with which it forms a salt bridge, are both in a buried environment as can be seen in e (the structural elements and density above the plane of the figure are not shown for clarity). Because there are no other basic residues near Glu1365 to neutralize its charge, the loss of the arginine charge in the R1285Q mutant would leave a net charge in a buried environment and destabilize the zipper. h, Mono view of the DNA density inside the ring, abutted by the zipper on the left side.

Extended Data Fig. 8 Cryo-EM reconstructions of the IDUb complex bound to ICL DNA, 5′-flap DNA and dsDNA, and of the IDnonUb:Ub complex ubiquitinated only on FANCD2 bound to nicked DNA.

a, Cryo-EM reconstruction of IDUb bound to ICL DNA (same DNA as Extended Data Fig. 2a). The consensus map (top), temperature-factor sharpened and masked, is coloured by local resolution estimated with the RELION3 postprocess program. The resolution range is mapped to the colours in the inset. The map is oriented as in Extended Data Fig. 7b. To make the DNA easier to see, the consensus map is also shown before temperature-factor sharpening with the FANCD2 portion above the plane of the figure clipped (middle, grey map) in the same orientation as Fig. 2c. The model shown is the IDUb-nicked DNA complex structure that was fit as a single body into the map with CHIMERA, without any further refinement of the coordinates. Because the DNA density in the consensus map appeared longer than the 20-bp dsDNA arms of the ICL DNA, we used 3D classification with partial signal subtraction followed by the reconstruction of the non-signal subtracted particles from four 3D classes (bottom row of four maps). This revealed that a 20-bp duplex arm goes in from either end of the clamp resulting in the consensus DNA density looking longer. In the first class, the DNA duplex reaches into the clamp from the FANCI side, and in the second class from the FANCD2 side. The third class seems to have mixed DNA registers with some bulbous density at the FANCD2 side and flat density at the FANCI side (the fourth class is devoid of DNA). This suggests that the ICL DNA has no preferred orientation in binding to IDUb. b, Cryo-EM reconstruction of IDUb bound to 5′-flap DNA containing two 29-bp duplexes flanking the flap (same DNA as the non-ubiquitinated complex of Extended Data Fig. 5b). The top and middle density are the consensus maps with and without temperature-factor sharpening, respectively, coloured as in a. The model shown is the IDUb-nicked DNA structure that was fit as a single body into the map. We did not refine the coordinates for this reconstruction, yet the DNA density overlaps well with the nicked DNA model. Unlike the ICL DNA complex, 3D classification failed to identify unique DNA conformations, and there was no evidence for any ssDNA branch (not shown). This suggests that either the flap is past the 29-bp duplex ends, or it can be accommodated within the clamp but not in a specific register that can be identified by 3D classification. c, Cryo-EM reconstruction of IDUb bound to 58-bp dsDNA, shown as in b. d, Gold-standard FSC plots for the consensus reconstructions of IDUb bound to the other DNA molecules discussed in the main text. The blue curve is the 3.8 Å reconstruction from 98,750 particles of IDUb bound to 5′-flap DNA, the red curve is the 3.8 Å reconstruction from 85,078 particles of IDUb bound to dsDNA, and the green curve is the 4.4 Å reconstruction from 28,519 particles of IDUb bound to ICL-DNA. e, Flow chart of single-particle cryo-EM data processing for the IDnonUb:Ub complex ubiquitinated only on FANCD2 bound to nicked DNA. Consensus (top) and focused maps, temperature-factor sharpened and masked, are coloured by local resolution estimated with the RELION3 postprocess program. The resolution range is mapped to the colours in the inset next to each map. f, The graph shows gold-standard FSC plots between two independently refined half-maps for the consensus reconstruction (blue curve) and the three focused maps. The FSC curve for the final model compared with the composite map is shown in black. The dashed line marks the FSC cutoff of 0.143. g, h, The structure of the singly monoubiquitinated IDnonUb:Ub is overall very similar to that of IDUb, and the two superimpose with a an r.m.s.d. of 0.57 Å in the positions of 2,429 common Cα atoms. There is only a small shift in the FANCD2 segment that interacts with the FANCI ubiquitin (UbI) as shown in the expanded view of h, in which the red arrows indicate the approximately 1.5 Å local motion that the FANCD2 helical repeats undergo when FANCI is also ubiquitinated.

Extended Data Fig. 9 Ubiquitination induces FANCD2 conformational changes associated with alternative FANCI–FANCD2 contacts; DNA binding activity of IDUb.

a, After ubiquitination, FANCI does not change substantially (the entire molecule can be superimposed with an r.m.s.d. of 1.6 Å in 1,132 Cα positions), but FANCD2 undergoes two changes. FANCD2 from the ID complex (salmon) is superimposed on that of IDUb (pink) by aligning the second half of their NTDs (residues 255–587; 1.8 Å r.m.s.d. for 308 Cα positions), a segment that changes little on ubiquitination. UbD2 that is covalently attached to FANCD2 is in red, and the UbI with which it packs in green. The N-terminal portion of the NTD, which rotates by 38° towards FANCI as a mostly rigid body (residues 45 to 254, 0.68 Å r.m.s.d. for 202 Cα), is approximately marked by a bracket. This rotation at the centre of the UbI binding site enables FANCD2 to better embrace UbI, and also to interact with FANCI (NTD–helical domain junction, residues 529–593), the latter involving similar residues on FANCD2 but mostly different ones on FANCI. Similarly marked is the helical domain (residues 604–928) that rotates relative to the NTD by 15°. Additional tilting of helices within the helical domain results in the CTD that follows (residues 929 to C termini also marked) being rotated by 20° relative to the invariant portion of the NTD. be, The FANCI and FANCD2 ubiquitin-binding structural elements are distinct from those of commonly occurring ubiquitin-binding domains. Superposition of the UbI (green) bound to FANCD2Ub (pink) on the ubiquitin (orange) bound to the dimeric Vps29 CUE domain (blue) from PDB: 1P3Q (b), the Cbl-b UBA domain (PDB: 2OOP) (c), the UBZ domain of Faap20 (PDB: 3WWQ) (d), and the UIM domain of Vps27 (PDB: 1Q0W) (e). The ubiquitin hydrophobic patch residues (Leu8, Ile44 and Val70) are shown in stick representation for both ubiquitin molecules in each figure. The blue sphere in d is the zinc atom of the UBZ domain. The orientation is similar to that of Fig. 4a. It has been suggested that FANCD2 shares sequence homology with the CUE domain28. Although the 47-residue region of proposed homology (residues 191–237) partially overlaps the Ub-binding site, its structure is unrelated to that of the CUE domain, and Ub binding by FANCD2 is distinct. f, Molecular surface coloured according to electrostatic potential calculated with PyMOL in the absence of DNA (coloured −5 to +5 kT blue to red), in the same view as Fig. 4d and with structural elements and surfaces above the DNA similarly clipped to reveal the DNA. As with the ID–ICL DNA complex, the FANCIUb groove partially encircles the DNA through basic and polar residues from α33b, α36b on one side of the groove and α40, α42 on the other. Near the middle of the DNA, however, the extension of α48 that results from coiling with α50 of FANCD2Ub (Fig. 3e) gives rise to a new semi-circular basic groove, between α46 and α48 on one side and α19b and α20 of the NTD on the other, into which dsDNA binds (Fig. 4d). This is associated with one of the two DNA bends, which redirects the duplex away from clashing with the new position of FANCD2. Thereafter, the second bend occurs as the dsDNA is redirected by the CTD of FANCD2, which uses the localized patch of α48 and α50 to bind to the duplex, analogously to the ID–ICL DNA complex. The side chains near the DNA, shown in Fig. 4d as sticks, are from FANCIUb residues Lys291 (α15), Lys397 (α19b), Ser411 (α20), Lys793, Thr794 (α33b), Lys898 (α36b), Lys980 (α40), Lys1026 (α42), Lys1164 (α46), and Thr1238, Arg1242, Arg1245 and Lys1248 on the extended α48; and from FANCD2Ub residues His1288, His1292, and Arg1299 on α48, and Thr1351, Arg1352 and Gln1355 on α50. The sites of DNA bending, of 26° and 31°, are centred on the 11th and 21st base pairs from the FANCI end, respectively, with large roll values over three base-pair steps. g, The IDUb Kd values for dsDNA, nicked, 5′-flap, ICL and fork DNA vary by less than a factor of two, with dsDNA and nicked DNA exhibiting slightly tighter binding. EMSA of the equimolar mixture of the monoubiquitinated FANCIUb and FANCD2Ub, each at the indicated concentrations, binding to the 32P-labelled DNA substrates (0.5 nM) shown schematically. The plots with a logarithmic x axis show the fraction bound in at least three repetitions of each experiment (different colours and shape markers) and their mean value (black dash). As with the non-ubiquitinated complex, the binding isotherms best fit a Hill slope model. A binding curve (black line) simulated with the indicated Kd and ηH values is shown on each plot.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics

Supplementary information

Supplementary Figure 1

This file contains the source data (autoradiograms, SDS-PAGE gels and immunoblots of gels) for the Main text and Extended Data figures.

Reporting Summary

Supplementary Table 1

This file contains the sequences of the oligonucleotides used to assemble the DNA substrates as indicated.

Supplementary Video 1 | Top mode of conformational flexibility of the FANCD2 CTD in the non-ubiquitinated ID-ICL DNA complex

The ten maps of the movie were generated by the relion_flex_analyze (RELION3) program for the top component from the PCA of the multi-body refinement angles described in the Extended Data Fig. 2d legend. The model from the consensus reconstruction is shown in cartoon representation and colored as in Fig. 1a.

Supplementary Video 2 | The second mode of conformational flexibility of the FANCD2 CTD in the non-ubiquitinated ID-ICL DNA complex

This video illustrates a second mode of conformational flexibility in the FANCD2 CTD of the non-ubiquitinated ID-ICL DNA complex. The ten maps are for the second PCA component (Extended Data Fig. 2e).

Supplementary Video 3 | Morph of the non-ubiquitinated ID changing to ubiquitinated IDUb

The FANCI and FANCD2 tails that are disordered in the ID complex were spliced on to it from the IDUb and flipped away from the structure. Other IDUb regions that undergo a disorder-to-order transition are deleted for morphing identical sets of amino acids. View looking down the rotation axis of the FANCD2 motion relative to FANCI.

Supplementary Video 4 | Morph from Supplementary Video 3 looking from the exterior bottom of the trough-like ID structure

Morph from Supplementary Video 3 looking from the exterior bottom of the trough-like ID structure.

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Wang, R., Wang, S., Dhar, A. et al. DNA clamp function of the monoubiquitinated Fanconi anaemia ID complex. Nature 580, 278–282 (2020). https://doi.org/10.1038/s41586-020-2110-6

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