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DNA clamp function of the monoubiquitinated Fanconi anaemia ID complex

Nature volume 580, pages 278–282 (2020)Cite this article

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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.

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Fig. 1: Human ID complex bound to ICL DNA.
Fig. 2: Monoubiquitinated human IDUb complex bound to nicked DNA.
Fig. 3: Conformational changes of ubiquitination.
Fig. 4: Interactions with ubiquitin and DNA.

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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.

References

  1. Ceccaldi, R., Sarangi, P. & D’Andrea, A. D. The Fanconi anaemia pathway: new players and new functions. Nat. Rev. Mol. Cell Biol. 17, 337–349 (2016).

    Article  CAS  Google Scholar 

  2. Zhang, J. et al. DNA interstrand cross-link repair requires replication-fork convergence. Nat. Struct. Mol. Biol. 22, 242–247 (2015).

    Article  CAS  Google Scholar 

  3. Boisvert, R. A. & Howlett, N. G. The Fanconi anemia ID2 complex: dueling saxes at the crossroads. Cell Cycle 13, 2999–3015 (2014).

    Article  CAS  Google Scholar 

  4. Park, W. H. et al. Direct DNA binding activity of the Fanconi anemia D2 protein. J. Biol. Chem. 280, 23593–23598 (2005).

    Article  CAS  Google Scholar 

  5. Yuan, F., El Hokayem, J., Zhou, W. & Zhang, Y. FANCI protein binds to DNA and interacts with FANCD2 to recognize branched structures. J. Biol. Chem. 284, 24443–24452 (2009).

    Article  CAS  Google Scholar 

  6. Longerich, S., San Filippo, J., Liu, D. & Sung, P. FANCI binds branched DNA and is monoubiquitinated by UBE2T–FANCL. J. Biol. Chem. 284, 23182–23186 (2009).

    Article  CAS  Google Scholar 

  7. Joo, W. et al. Structure of the FANCI-FANCD2 complex: insights into the Fanconi anemia DNA repair pathway. Science 333, 312–316 (2011).

    Article  ADS  CAS  Google Scholar 

  8. Raschle, M. et al. Mechanism of replication-coupled DNA interstrand crosslink repair. Cell 134, 969–980 (2008).

    Article  CAS  Google Scholar 

  9. Wang, R. et al. Mechanism of DNA interstrand cross-link processing by repair nuclease FAN1. Science 346, 1127–1130 (2014).

    Article  ADS  CAS  Google Scholar 

  10. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

    Article  Google Scholar 

  11. Nakane, T., Kimanius, D., Lindahl, E. & Scheres, S. H. Characterisation of molecular motions in cryo-EM single-particle data by multi-body refinement in RELION. eLife 7, e36861 (2018).

    Article  Google Scholar 

  12. Brown, A. et al. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. D 71, 136–153 (2015).

    Article  CAS  Google Scholar 

  13. Howlett, N. G., Taniguchi, T., Durkin, S. G., D’Andrea, A. D. & Glover, T. W. The Fanconi anemia pathway is required for the DNA replication stress response and for the regulation of common fragile site stability. Hum. Mol. Genet. 14, 693–701 (2005).

    Article  CAS  Google Scholar 

  14. Smogorzewska, A. et al. Identification of the FANCI protein, a monoubiquitinated FANCD2 paralog required for DNA repair. Cell 129, 289–301 (2007).

    Article  CAS  Google Scholar 

  15. Sirbu, B. M. et al. Identification of proteins at active, stalled, and collapsed replication forks using isolation of proteins on nascent DNA (iPOND) coupled with mass spectrometry. J. Biol. Chem. 288, 31458–31467 (2013).

    Article  CAS  Google Scholar 

  16. Yates, M. & Maréchal, A. Ubiquitylation at the fork: making and breaking chains to complete DNA replication. Int. J. Mol. Sci. 19, 2909 (2018).

    Article  Google Scholar 

  17. Aymami, J. et al. Molecular structure of nicked DNA: a substrate for DNA repair enzymes. Proc. Natl Acad. Sci. USA 87, 2526–2530 (1990).

    Article  ADS  CAS  Google Scholar 

  18. Ho, P. S. Structure of the Holliday junction: applications beyond recombination. Biochem. Soc. Trans. 45, 1149–1158 (2017).

    Article  CAS  Google Scholar 

  19. Sato, K., Toda, K., Ishiai, M., Takata, M. & Kurumizaka, H. DNA robustly stimulates FANCD2 monoubiquitylation in the complex with FANCI. Nucleic Acids Res. 40, 4553–4561 (2012).

    Article  CAS  Google Scholar 

  20. Longerich, S. et al. Regulation of FANCD2 and FANCI monoubiquitination by their interaction and by DNA. Nucleic Acids Res. 42, 5657–5670 (2014).

    Article  CAS  Google Scholar 

  21. Rajendra, E. et al. The genetic and biochemical basis of FANCD2 monoubiquitination. Mol. Cell 54, 858–869 (2014).

    Article  CAS  Google Scholar 

  22. Colnaghi, L. et al. Patient-derived C-terminal mutation of FANCI causes protein mislocalization and reveals putative EDGE motif function in DNA repair. Blood 117, 2247–2256 (2011).

    Article  CAS  Google Scholar 

  23. Husnjak, K. & Dikic, I. Ubiquitin-binding proteins: decoders of ubiquitin-mediated cellular functions. Annu. Rev. Biochem. 81, 291–322 (2012).

    Article  CAS  Google Scholar 

  24. Yamamoto, K. N. et al. Involvement of SLX4 in interstrand cross-link repair is regulated by the Fanconi anemia pathway. Proc. Natl Acad. Sci. USA 108, 6492–6496 (2011).

    Article  ADS  CAS  Google Scholar 

  25. Lachaud, C. et al. Distinct functional roles for the two SLX4 ubiquitin-binding UBZ domains mutated in Fanconi anemia. J. Cell Sci. 127, 2811–2817 (2014).

    Article  CAS  Google Scholar 

  26. Montes de Oca, R. et al. Regulated interaction of the Fanconi anemia protein, FANCD2, with chromatin. Blood 105, 1003–1009 (2005).

    Article  Google Scholar 

  27. Ishiai, M. et al. FANCI phosphorylation functions as a molecular switch to turn on the Fanconi anemia pathway. Nat. Struct. Mol. Biol. 15, 1138–1146 (2008).

    Article  CAS  Google Scholar 

  28. Rego, M. A., Kolling, F. W., IV, Vuono, E. A., Mauro, M. & Howlett, N. G. Regulation of the Fanconi anemia pathway by a CUE ubiquitin-binding domain in the FANCD2 protein. Blood 120, 2109–2117 (2012).

    Article  CAS  Google Scholar 

Download references

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.

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Authors and Affiliations

  1. Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

    Renjing Wang, Shengliu Wang, Ankita Dhar, Christopher Peralta & Nikola P. Pavletich

  2. Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Shengliu Wang, Christopher Peralta & Nikola P. Pavletich

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  1. Renjing Wang
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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.

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Correspondence to Nikola P. Pavletich.

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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. b–e, 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
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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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