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

Nature Structural & Molecular Biology volume 27, pages 240–248 (2020)Cite this article

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Abstract

Vertebrate DNA crosslink repair excises toxic replication-blocking DNA crosslinks. Numerous factors involved in crosslink repair have been identified, and mutations in their corresponding genes cause Fanconi anemia (FA). A key step in crosslink repair is monoubiquitination of the FANCD2–FANCI heterodimer, which then recruits nucleases to remove the DNA lesion. Here, we use cryo-EM to determine the structures of recombinant chicken FANCD2 and FANCI complexes. FANCD2–FANCI adopts a closed conformation when the FANCD2 subunit is monoubiquitinated, creating a channel that encloses double-stranded DNA (dsDNA). Ubiquitin is positioned at the interface of FANCD2 and FANCI, where it acts as a covalent molecular pin to trap the complex on DNA. In contrast, isolated FANCD2 is a homodimer that is unable to bind DNA, suggestive of an autoinhibitory mechanism that prevents premature activation. Together, our work suggests that FANCD2–FANCI is a clamp that is locked onto DNA by ubiquitin, with distinct interfaces that may recruit other DNA repair factors.

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Fig. 1: Purification and structures of D2–I and ubD2–I.
Fig. 2: Ubiquitin is anchored to K563 on FANCD2 but also contacts FANCI.
Fig. 3: ubD2–I is a DNA clamp.
Fig. 4: Monoubiquitination locks the D2–I clamp onto DNA.
Fig. 5: FANCI forms a monomer and binds DNA, whereas FANCD2 is dimeric and does not bind DNA.

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Data availability

Cryo-EM maps generated during this study have been deposited in the Electron Microscopy Data Bank (EMDB) with accession codes EMD-10532 (D2–I), EMD-10531 (ubD2–I), and EMD-10534 (D2–D2). Models generated during this study have been deposited in the protein databank (PDB) with accession codes PDB 6TNG (D2–I), PDB 6TNF (ubD2–I) and PDB 6TNI (D2–D2). MS data have been deposited in the PRIDE database with accession code PXD017020. Original gels and blots in Figs. 1b, 4 and 5b and Extended Data Figs. 1b, 3b,c, 4a,b and 5 are provided in Supplementary Fig. 1. Data for quantifications in Fig. 4 and Extended Data Fig. 4c are available as Source data with the paper online.

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Acknowledgements

We are grateful to T. Nakane, S. H. W. Scheres, F. O’Reilly, M. Babu, P. Emsley, J. Pruneda, T. Sijacki, J. A. W. Stowell and members of the Passmore lab for assistance and advice; the LMB EM facility, J. Grimmett and T. Darling (LMB scientific computation), and J. G. Shi (baculovirus) for support. This work was supported by the Medical Research Council, as part of United Kingdom Research and Innovation, MRC file reference numbers MC_U105192715 (L.A.P.) and MC_U105178811 (K.J.P.), and the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) grant no. 329673113 (J.R.). The Wellcome Centre for Cell Biology is supported by core funding from the Wellcome Trust (grant no. 203149; J.R.). P.A. is supported by an EMBO Long-Term Fellowship (ALTF 692–2018). We acknowledge Diamond Light Source for access to eBIC (proposals EM17434 and BI23268) funded by the Wellcome Trust, MRC and Biotechnology and Biological Sciences Research Council.

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Author notes

  1. These authors contributed equally: Pablo Alcón, Shabih Shakeel.

Authors and Affiliations

  1. MRC Laboratory of Molecular Biology, Cambridge, UK

    Pablo Alcón, Shabih Shakeel, Ketan J. Patel & Lori A. Passmore

  2. Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany

    Zhuo A. Chen & Juri Rappsilber

  3. Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK

    Juri Rappsilber

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Contributions

P.A. and S.S. designed protein expression and purification schemes, performed ubiquitination and binding assays and performed cryo-EM, 3D reconstruction and modeling; Z.A.C. and J.R. performed crosslinking MS analysis. L.A.P. and K.J.P. supervised the research; all authors contributed to writing the paper.

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Correspondence to Lori A. Passmore.

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Peer review information Anke Sparmann and Inês Chen were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Purification of FANCI, FANCD2 and D2–I, and cryo-EM of D2–I, ubD2–I and D2 dimer.

a, Coomassie gel showing purified His-tagged FANCI, StrepII-tagged FANCD2 and D2–I after gel filtration. b–e, Cryo-EM of D2–I, ubD2–I and D2. b, Representative micrographs. Selected individual particles are marked with green circles. Scale bars, 25 nm. c, Fourier shell correlation (FSC) curves for gold-standard refinements. d, Angular distribution density plots of particles used in 3D reconstructions calculated using cryoEF63. Every point is a particle orientation, and the color scale represents the normalized density of views around this point. The color scale runs from 0 (low, blue) to 0.00026 (high, red). All complexes had a preferred orientation. Note that C2 symmetry was applied for FANCD2. e, Local resolution estimates calculated using ResMap64. Uncropped image for a is available in Supplementary Fig. 1.

Extended Data Fig. 2 Model fitting.

a, Overall fit of model to map for ubD2–I, D2–I and D2 (blue, FANCD2; magenta, FANCI; green, ubiquitin; yellow, DNA). b, Representative fits of model to map for FANCD2, FANCI, DNA and ubiquitin in the ubD2–I structure. c, FSC curves for model versus map. d, FANCD2 and FANCI structures from G. gallus (gg) were aligned with each other and with the M. musculus (mm)31 crystal structures using PDBeFOLD62 (http://www.ebi.ac.uk/msd-srv/ssm/); figures were prepared with PyMOL (The PyMOL Molecular Graphics System, Version 2.0, Schrödinger, LLC; https://pymol.org/2/).

Extended Data Fig. 3 Crosslinking mass spectrometry and analysis of DNA binding by FANCD2 and FANCI.

a, Distribution histogram of Cα−Cα distances between linked residue pairs in the 3D model of ubD2–I (left). Crosslinks with Cα−Cα distances below the theoretical crosslinking limit (30 Å) are shown in green. Overlength crosslinks (>30 Å) are shown in red. The distribution of Cα−Cα distances between random crosslinkable residue pairs in the 3D model is shown in gray. Crosslinks mapped onto the front view of the 3D structure are shown on the right. b, Monoubiquitination assays were assembled in the presence of 5 μM linear dsDNA of differing lengths (10–44 bp). DNA binding was analyzed by EMSA (top) after loading the reactions onto native gels and imaging of the fluorescently labeled DNA. Monoubiquitination efficiency was analyzed by Coomassie blue (middle) and western blotting the His-tagged ubiquitin (bottom). Controls lacking ubiquitin or DNA are indicated. These data are representative of experiments performed three times. c, Monoubiquitination assays were assembled without (left) and with (right) ubiquitin, both in the presence of a 39-bp dsDNA at 100 nM and increasing amounts of D2–I (0–1,000 nM). Assays were analyzed by EMSA (top, imaging for the fluorescently labeled DNA) or western blotting His-tagged ubiquitin (bottom). We cannot exclude that other proteins may interact with DNA in these assays, but the migration positions of the shifted bands are similar to the experiment in Fig. 4, in which ubD2–I was purified away from all other proteins. These data are representative of experiments performed three times. Uncropped images for b,c are available in Supplementary Fig. 1.

Extended Data Fig. 4 FANCD2 and FANCI oligomerization state and DNA binding activity.

a, Size-exclusion chromatogram as shown in Fig. 5a (top). Peak fractions were analyzed via SDS−PAGE (bottom). A single asterisk (*) indicates the migration position for monomers. A double asterisk (**) indicates the migration position for dimers. These results are representative of experiments performed three times. b, DNA binding of FANCI, FANCD2, D2–I and FANCI mixed with FANCD2 was analyzed in EMSAs performed with 20 nM 39-bp dsDNA and 0–140 nM protein. Gels shown are representative gels of experiments performed independently three times. The FANCD2 and D2–I gels are same as in Fig 5b. c, Quantification of mean intensities of free DNA from b. Error bars represent the standard deviation. Individual data points (n = 3 independent experiments) are shown. The means are connected by lines for clarity. Uncropped images for a,b are available in Supplementary Fig. 1. Data for the plot in c are available as Source data.

Source data

Extended Data Fig. 5 FANCD2 dimers cannot be ubiquitinated but they exchange with FANCI to form a D2–I heterodimer.

a, Monoubiquitination assays of D2–I and FANCD2 homodimer. The FANCD2 homodimer had a StrepII-tag and was therefore larger than D2 in the D2–I complex. Monoubiquitination efficiency was analyzed by Coomassie blue SDS−PAGE (top) and western blotting the His-tagged ubiquitin (bottom). b, Exchange assay. The FANCD2 homodimer was immobilized on Strep-Tactin resin and incubated with free FANCI. The resin was washed, then bound and unbound fractions were analyzed via by SDS−PAGE. These data are representative of experiments performed twice. Uncropped images are available in Supplementary Fig. 1.

Supplementary information

Supplementary Information

Supplementary Notes 1 and 2, Supplementary Tables 1 and 3 and Supplementary Fig. 1.

Reporting Summary

Supplementary Video 1

Motions detected by multibody refinement of ubD2–I: principal component analysis of multibody refinement results revealed two major motions.

Supplementary Video 2

Morph between D2–I and ubD2–I structures: a morph between the unmodified and modified D2–I complexes shows how FANCD2 and FANCI rotate around a hinge to clamp around DNA. In this video, the complexes are viewed from the back side of the hinge where ubiquitin is located.

Supplementary Video 3

Morph between D2–I and ubD2–I structures: a morph between the unmodified and modified D2–I complexes shows how FANCD2 and FANCI rotate around a hinge to clamp around DNA. In this video, the complexes are viewed from the DNA-binding groove.

Supplementary Video 4

Morph between D2–I and ubD2–I structures: a morph between the unmodified and modified D2–I complexes shows how FANCD2 and FANCI rotate around a hinge to clamp around DNA. In this video, the complexes are viewed from the top.

Supplementary Table 2

Crosslinking mass spectrometry data file.

Source data

Source Data Fig. 4

Statistical source data

Source Data Extended Data Fig. 4

Statistical source data

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Alcón, P., Shakeel, S., Chen, Z.A. et al. FANCD2–FANCI is a clamp stabilized on DNA by monoubiquitination of FANCD2 during DNA repair. Nat Struct Mol Biol 27, 240–248 (2020). https://doi.org/10.1038/s41594-020-0380-1

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