Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

The DNA-damage kinase ATR activates the FANCD2-FANCI clamp by priming it for ubiquitination

Nature Structural & Molecular Biology volume 29, pages 881–890 (2022)Cite this article

Subjects

Abstract

DNA interstrand cross-links are tumor-inducing lesions that block DNA replication and transcription. When cross-links are detected at stalled replication forks, ATR kinase phosphorylates FANCI, which stimulates monoubiquitination of the FANCD2–FANCI clamp by the Fanconi anemia core complex. Monoubiquitinated FANCD2–FANCI is locked onto DNA and recruits nucleases that mediate DNA repair. However, it remains unclear how phosphorylation activates this pathway. Here, we report structures of FANCD2–FANCI complexes containing phosphomimetic FANCI. We observe that, unlike wild-type FANCD2–FANCI, the phosphomimetic complex closes around DNA, independent of the Fanconi anemia core complex. The phosphomimetic mutations do not substantially alter DNA binding but instead destabilize the open state of FANCD2–FANCI and alter its conformational dynamics. Overall, our results demonstrate that phosphorylation primes the FANCD2–FANCI clamp for ubiquitination, showing how multiple posttranslational modifications are coordinated to control DNA repair.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Phosphomimetic mutations of conserved ATR phosphorylation sites in the FANCI phospho-loop stimulate monoubiquitination of FANCD2.
Fig. 2: Phosphomimetic D2-I3D closes around DNA.
Fig. 3: Closed, DNA-bound D2-I3D is poised for monoubiquitination.
Fig. 4: D2-IWT, D2-I3A and D2-I3D exhibit similar DNA-binding kinetics.
Fig. 5: Phosphomimetic mutations result in exposure of target lysines in D2-I.
Fig. 6: Local rearrangements at the heterodimerization interface promote closure of D2-I3D.
Fig. 7: Model of FA pathway activation through FANCI phosphorylation.

Similar content being viewed by others

Data availability

Cryo-EM maps have been deposited in the EM Data Bank with the following accession codes: EMD-15102 (D2-I3D), EMD-15101 (DNA-D2-I3D) and EMD-15103 (DNA-ubD2-I3D). Atomic coordinates of DNA-D2-I3D have been deposited in the Protein Data Bank with the accession code PDB 8A2Q. MS data have been deposited in jPOST (project ID JPST001474, PRIDE ID: PXD031632)54. Source data are provided with this paper. All other data are available in the main text or as part of the Extended Data or supplementary materials. Original gels, blot images and numerical data used to generate plots are provided in the source data. Correspondence and requests for materials should be addressed to L.A.P. All unique materials are available upon request with completion of a standard Materials Transfer Agreement.

References

  1. Knipscheer, P. et al. The Fanconi anemia pathway promotes replication-dependent DNA interstrand cross-link repair. Science 326, 1698–1701 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Semlow, D. R. & Walter, J. C. Mechanisms of vertebrate DNA interstrand cross-link repair. Annu. Rev. Biochem. 90, 107–135 (2021).

    Article  CAS  PubMed  Google Scholar 

  4. Crossan, G. P. & Patel, K. J. The Fanconi anaemia pathway orchestrates incisions at sites of crosslinked DNA. J. Pathol. 226, 326–337 (2012).

    Article  CAS  PubMed  Google Scholar 

  5. Walden, H. & Deans, A. J. The Fanconi anemia DNA repair pathway: structural and functional insights into a complex disorder. Annu. Rev. Biophys. 43, 257–278 (2014).

    Article  CAS  PubMed  Google Scholar 

  6. Alpi, A. et al. UBE2T, the Fanconi anemia core complex, and FANCD2 are recruited independently to chromatin: a basis for the regulation of FANCD2 monoubiquitination. Mol. Cell. Biol. 27, 8421–8430 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Garcia-Higuera, I. et al. Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway. Mol. Cell 7, 249–262 (2001).

    Article  CAS  PubMed  Google Scholar 

  8. Machida, Y. J. et al. UBE2T is the E2 in the Fanconi anemia pathway and undergoes negative autoregulation. Mol. Cell 23, 589–596 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Meetei, A. R. et al. A novel ubiquitin ligase is deficient in Fanconi anemia. Nat. Genet. 35, 165–170 (2003).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Klein Douwel, D. et al. XPF-ERCC1 acts in unhooking DNA interstrand crosslinks in cooperation with FANCD2 and FANCP/SLX4. Mol. Cell 54, 460–471 (2014).

    Article  CAS  PubMed  Google Scholar 

  12. Taniguchi, T. et al. S-phase-specific interaction of the Fanconi anemia protein, FANCD2, with BRCA1 and RAD51. Blood 100, 2414–2420 (2002).

    Article  CAS  PubMed  Google Scholar 

  13. Kim, J. M. et al. Inactivation of murine Usp1 results in genomic instability and a Fanconi anemia phenotype. Dev. Cell 16, 314–320 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Nijman, S. M. et al. The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway. Mol. Cell 17, 331–339 (2005).

    Article  CAS  PubMed  Google Scholar 

  15. Oestergaard, V. H. et al. Deubiquitination of FANCD2 is required for DNA crosslink repair. Mol. Cell 28, 798–809 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Alcon, P. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Rennie, M. L., Arkinson, C., Chaugule, V. K., Toth, R. & Walden, H. Structural basis of FANCD2 deubiquitination by USP1-UAF1. Nat. Struct. Mol. Biol. 28, 356–364 (2021).

    Article  CAS  PubMed  Google Scholar 

  19. Rennie, M. L. et al. Differential functions of FANCI and FANCD2 ubiquitination stabilize ID2 complex on DNA. EMBO Rep. 21, e50133 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Shakeel, S. et al. Structure of the Fanconi anaemia monoubiquitin ligase complex. Nature 575, 234–237 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Tan, W. et al. Monoubiquitination by the human Fanconi anemia core complex clamps FANCI:FANCD2 on DNA in filamentous arrays. eLife 9, e54128 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Wang, R., Wang, S., Dhar, A., Peralta, C. & Pavletich, N. P. DNA clamp function of the monoubiquitinated Fanconi anaemia ID complex. Nature 580, 278–282 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wang, S., Wang, R., Peralta, C., Yaseen, A. & Pavletich, N. P. Structure of the FA core ubiquitin ligase closing the ID clamp on DNA. Nat. Struct. Mol. Biol. 28, 300–309 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 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  PubMed  PubMed Central  Google Scholar 

  26. van Twest, S. et al. Mechanism of ubiquitination and deubiquitination in the Fanconi anemia pathway. Mol. Cell 65, 247–259 (2017).

    Article  PubMed  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  PubMed  PubMed Central  Google Scholar 

  28. Andreassen, P. R., D’Andrea, A. D. & Taniguchi, T. ATR couples FANCD2 monoubiquitination to the DNA-damage response. Genes Dev. 18, 1958–1963 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Cheung, R. S. et al. Ubiquitination-linked phosphorylation of the FANCI S/TQ cluster contributes to activation of the Fanconi anemia I/D2 complex. Cell Rep. 19, 2432–2440 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Shigechi, T. et al. ATR-ATRIP kinase complex triggers activation of the Fanconi anemia DNA repair pathway. Cancer Res. 72, 1149–1156 (2012).

    Article  CAS  PubMed  Google Scholar 

  31. Tan, W., van Twest, S., Murphy, V. J. & Deans, A. J. ATR-mediated FANCI phosphorylation regulates both ubiquitination and deubiquitination of FANCD2. Front. Cell Dev. Biol. 8, 2 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Ishiai, M. et al. Activation of the FA pathway mediated by phosphorylation and ubiquitination. Mutat. Res. 803–805, 89–95 (2017).

    Article  PubMed  Google Scholar 

  33. Chaugule, V. K., Arkinson, C., Toth, R. & Walden, H. Enzymatic preparation of monoubiquitinated FANCD2 and FANCI proteins. Methods Enzymol. 618, 73–104 (2019).

    Article  CAS  PubMed  Google Scholar 

  34. Russo, C. J. & Passmore, L. A. Electron microscopy: ultrastable gold substrates for electron cryomicroscopy. Science 346, 1377–1380 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

  36. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Wagner, T. et al. SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM. Commun. Biol. 2, 218 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Tan, Y. Z. et al. Addressing preferred specimen orientation in single-particle cryo-EM through tilting. Nat. Methods 14, 793–796 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  42. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Sanchez-Garcia, R. et al. DeepEMhancer: a deep learning solution for cryo-EM volume post-processing. Commun. Biol. 4, 874 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  46. Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).

    Article  CAS  PubMed  Google Scholar 

  47. Croll, T. I. ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallogr. D Struct. Biol. 74, 519–530 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Barad, B. A. et al. EMRinger: side chain-directed model and map validation for 3D cryo-electron microscopy. Nat. Methods 12, 943–946 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Chen, Z. A. et al. Architecture of the RNA polymerase II-TFIIF complex revealed by cross-linking and mass spectrometry. EMBO J. 29, 717–726 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896–1906 (2007).

    Article  CAS  PubMed  Google Scholar 

  51. Mendes, M. L. et al. An integrated workflow for crosslinking mass spectrometry. Mol. Syst. Biol. 15, e8994 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Fischer, L. & Rappsilber, J. Quirks of error estimation in cross-linking/mass spectrometry. Anal. Chem. 89, 3829–3833 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Chen, Z. A. & Rappsilber, J. Quantitative cross-linking/mass spectrometry to elucidate structural changes in proteins and their complexes. Nat. Protoc. 14, 171–201 (2019).

    Article  PubMed  Google Scholar 

  54. Okuda, S. et al. jPOSTrepo: an international standard data repository for proteomes. Nucleic Acids Res. 45, D1107–D1111 (2017).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful to C. Johnson, K. Nguyen, R. Williams, K. J. Patel and members of the Passmore laboratory for assistance and advice; the MRC-LMB EM facility for access and support with EM sample preparation and data collection; J. Rodriguez Molina and V. Chandrasekaran (MRC-LMB) for assisting with cryo-EM data collection; J. Grimmett and T. Darling (MRC-LMB scientific computation); and J.G. Shi (MRC-LMB baculovirus) for support. We acknowledge Diamond Light Source for access to eBIC (proposal BI23268) funded by the Wellcome Trust, MRC and Biotechnology and Biological Sciences Research Council. This work was supported by the MRC as part of UK Research and Innovation, MRC file reference number MC_U105192715 (L.A.P.); a PhD studentship from the Cambridge Trust (T.S.); an EMBO Long-Term Fellowship, grant ALTF 692-2018 (P.A.); and the Deutsche Forschungsgemeinschaft (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.).

Author information

Author notes

  1. Shabih Shakeel

    Present address: Walter and Eliza Hall Institute of Medical Research, Parkville, Australia

  2. These authors contributed equally: Pablo Alcón, Zhuo A. Chen.

Authors and Affiliations

  1. MRC Laboratory of Molecular Biology, Cambridge, UK

    Tamara Sijacki, Pablo Alcón, Stephen H. McLaughlin, Shabih Shakeel & Lori A. Passmore

  2. Technische Universität Berlin, Chair of Bioanalytics, Berlin, Germany

    Zhuo A. Chen & Juri Rappsilber

Authors
  1. Tamara Sijacki
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pablo Alcón
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhuo A. Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stephen H. McLaughlin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Shabih Shakeel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Juri Rappsilber
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lori A. Passmore
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.S., P.A. and S.S. designed protein purification schemes and expressed and purified proteins. T.S. generated constructs, carried out ubiquitination assays and together with S.H.M. performed DNA binding studies. T.S. prepared samples for cryo-EM and collected data with S.S. T.S. and P.A. processed cryo-EM data. Z.A.C. and J.R. performed cross-linking MS analysis. L.A.P. supervised the research. T.S. and L.A.P. wrote the manuscript, with contributions from all authors.

Corresponding author

Correspondence to Lori A. Passmore.

Ethics declarations

Competing interests

L.A.P. is an inventor on a patent filed by the Medical Research Council for all-gold EM supports, licensed to Quantifoil under the trademark UltrAuFoil. The remaining authors declare no competing interests.

Peer review

Peer review information

Nature Structural & Molecular Biology thanks Patrick Sung, Nicolas Thoma and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editors: Beth Moorefield and Carolina Perdigoto, in collaboration with the Nature Structural & Molecular Biology team.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Phosphomimetic mutations in the phospho-loop of FANCI stimulate FANCD2 monoubiquitination.

(a) Surface representation of the human closed D2-I complex bound to the FA core complex and DNA (PDB 7KZV), from which only FANCD2 (blue) and FANCI (magenta) are shown. The position of the phospho-loop (orange dashed line; residues 550–576 in human, 550–583 in chicken) is shown relative to the D2-I ubiquitination sites FANCD2-K563 and FANCI-K525 (green), as well as the interaction interfaces of the D2-I complex with: the FA core complex E3 ligase subunit FANCL (red, PDB 7KZV), the E2 ubiquitin conjugating enzyme Ube2T (purple, PDB 7KZV), the FA core complex subunit FANCE (dark blue, PDB 7KZV) and UAF1 (salmon, PDB 7AY1). (b) Coomassie-stained SDS-PAGE of purified 6xHis-tagged FANCIWT, FANCI3D, FANCI3A and 2xStrepII-tagged FANCD2 after gel filtration. Purifications have been reproduced independently twice (FANCIWT), or four times (FANCI3D and FANCD2) with similar results. FANCI3A was purified only once, since the yield was sufficient to carry out all the performed assays. (c) Control reactions of monoubiquitination assays (shown in Fig. 1b) carried out for 20 minutes. Two independently performed assays were carried out and showed the same result.

Source data

Extended Data Fig. 2 CryoEM analysis of DNA-free, DNA-bound and ubiquitinated D2-I3D.

(a) Representative micrographs and selected 2D class-averages from the D2-I3D (left, dataset of 1,845 micrographs), DNA-bound D2-I3D (middle, dataset of 14,842 micrographs) and DNA-bound ubiquitinated D2-I3D (right, dataset of 6,515 micrographs) datasets (top). (b) Fourier-shell correlation (FSC) curves of the final consensus reconstructions. For the DNA-bound D2-I3D dataset, FSC curves of individual FANCD2 and FANCI maps, obtained after signal subtraction and refinement, are also shown. Reported resolutions were determined at FSC = 0.143. (c) Directional FSC plots and sphericity values calculated using the 3D-FSC server. The D2-I3D map has a strongly preferred orientation. (d) Local resolutions (obtained in Relion3.1) plotted on the final maps. N- and C-termini of both proteins are indicated (N and C, in pink for FANCI and in blue for FANCD2). The C-terminal region of FANCI and both the N- and the C-terminal regions of FANCD2 in D2-I3D are poorly defined in the open conformation in the absence of DNA, suggesting that they are flexible. These regions are better defined in the maps of DNA-bound D2-I3D and ubD2-I3D, consistent with them becoming more ordered after clamping onto DNA.

Extended Data Fig. 3 Cryo-EM processing pipeline and modelling of DNA-bound D2-I3D.

(a) CryoEM processing pipeline for the DNA-D2-I3D complex. A similar pipeline was used to process the D2-I3D and ubD2-I3D datasets. A crYOLO model was trained on a subset of micrographs and then used to pick all particles. An ab initio model was obtained after particle extraction and 2D classification. Several rounds of 3D refinement and classification, followed by CTF refinement and Bayesian polishing were performed next. The final consensus map was obtained after refinement of two selected classes (with the best density for the N-terminal region of FANCD2 and DNA) resulting from a 3D classification without image alignment. Individual maps of FANCD2 and FANCI were obtained by signal subtraction of the FANCI-DNA and the FANCD2-DNA density respectively, followed by a 3D refinement. Sharpened maps (B factor of -55 for FANCD2 and -60 for FANCI) were used for interpretation and modelling. (b) Modelling of FANCD2 and FANCI. Fit of the D2-I complex (shown as sticks) in the composite map (sharpened using B factor of -55). (c) Fit of the FANCD2 and FANCI models (shown as sticks) into the corresponding signal subtracted and focus-refined maps (sharpened using B factor of either -55 or -60). Representative densities of the C-terminal and the N-terminal regions of either FANCD2 or FANCI from the DNA-D2-I3D (sharpened map) and the corresponding model regions (cartoon representation, side chains represented as sticks (oxygen atoms in red, nitrogen in blue) are shown. (d) A bent dsDNA was fitted into the corresponding DNA density (postprocessed composite map on the left and unsharpened map of DNA on the right). (e) Comparison of the FANCI and FANCD2 chains from the DNA-D2-I3D model to the corresponding FANCI and FANCD2 chains from the G. gallus D2-IWT (PDB 6TNG) and the ubiquitinated D2-IWT (PDB 6TNF) complexes.

Extended Data Fig. 4 DNA binding of D2-I3D is similar to that of D2-IWT.

(a) Quantification of EMSAs shown in Fig. 4a. Data obtained from three independently performed assays was quantified as percentage of free DNA. Data points of each replicate (symbols) were plotted and fit to a one step exponential decay as described in the Methods. (b) EMSAs performed in buffers with different NaCl concentrations (75, 300, 400 and 500 mM). (c) Quantification of the EMSA salt titration series from panel (b). Data points of three independently performed assays representing the percentage of free DNA at increasing protein concentrations were plotted and fitted as above.

Source data

Extended Data Fig. 5 Charges within the phospho-loop destabilize the open conformation of D2-I.

(a) Surface representation of the M. musculus crystal structure of D2-IWT (PDB 3S4W, FANCD2 in blue, FANCI in magenta), in which the phospho-loop was modelled at the dimerization interface of the open D2-I complex (left). The electrostatic potential of the phospho-loop region (calculated in ChimeraX) is shown. The scale used to represent the electrostatic potential is a gradient from −10 (red) to +10 (blue) kT, and regions with a net charge of 0 are colored in white. The surface to which the three S/TQ motifs (ATR phosphorylation sites) map to is mostly neutral, whereas the proximal FANCD2 surface exhibits negative electrostatic potential (middle). The introduction of bulky, charged groups could cause steric or charge-based repulsions that disrupt FANCI interaction with the negatively-charged surface of FANCD2 at the interface. A zoomed-in view of this interface is shown on the right (cartoon representation) where selected residues (sticks) and their H-bonding network (dashed blue lines, generated in ChimeraX) are depicted. The three ATR target residues (yellow) in the phospho-loop (orange) along with other selected residues (tan) are shown as sticks. (b-d) Control reactions of the 160-minute time-point of the monoubiquitination assay shown in Fig. 5a-c performed in the absence of DNA (b), presence of 100 nM dsDNA (c), or presence of 5 µM 44-bp dsDNA (d). Two independently performed assays have been carried out and showed the same result. FA-CC, FA core complex.

Source data

Extended Data Fig. 6 Monoubiquitination assays in the absence of DNA suggest that the three phosphomimetic mutations have an additive effect on D2-I closure.

(a) Time-course monoubiquitination assays in the absence of DNA were performed with FANCD2 incubated with different FANCI constructs and analyzed using SDS-PAGE. Eight different FANCI constructs were used: wild-type FANCI, FANCI-Dxx (S558D), FANCI-xDx (S561D), FANCI-xxD (T567D), FANCI-DDx (S558D and S561D), FANCI-DxD (S558D and T567D), FANCI-xDD (S561D and T567D), FANCI-3D (S558D, S561D and T567D), and FANCI-delta loop (deletion of residues 555–582). (b) Quantification of monoubiquitination assays performed in panel a. Data obtained from four independent assays were quantified as percentage of ubiquitinated FANCD2. Replicate data were fitted using a global non-linear regression function and plotted. Data points of each replicate (symbols), the corresponding means (solid lines) and the 95% confidence intervals (shaded area between dashed lines) are shown.

Source data

Extended Data Fig. 7 Quantitative crosslinking mass spectrometry of D2-IWT and D2-I3D performed in the presence and absence of DNA.

(a) Maps of crosslinks identified in the four D2-I complexes (3% FDR at residue pair level): D2-IWT (top, left), D2-I3D (top, right), D2-IWT with 44-bp dsDNA (bottom, left) and D2-I3D with 44-bp dsDNA (bottom, right). Proteins are shown as bars (FANCD2 in blue and FANCI in magenta with their N- and C-termini labeled). A subset of crosslinks discussed in the main text is shown in different colors, while the rest is colored in black. Crosslinks only identified in the D2-IWT (green lines) and a cluster of crosslinks between FANCI and N-FANCD2 which is enriched in D2-I3D, but also identified in DNA-bound D2-IWT and DNA-bound D2-I3D (orange lines) are shown. Crosslinks at the C-terminal dimerization interface of FANCD2 and FANCI that are only observed in DNA-bound D2-IWT and DNA-bound D2-I3D are also shown (magenta lines). (b) Close-up view of the region in the crosslink maps of all four samples containing the two clusters that indicate a change in position of the FANCD2 NTR. Some crosslinks in D2-IWT disappear in the other three samples (D2-I3D, DNA-bound D2-IWT and DNA-bound D2-I3D) (green lines; NTR crosslinks encircled with a red line), and instead a new cluster of crosslinks is identified (orange lines; NTR corsslinks encircled with a black line). Grey lines indicate other identified crosslinks. (c) Quantified crosslinks (FANCI-524 to FANCD2–253 and FANCI-529 to FANCD2-291, yellow lines) in proximity of FANCI-K525 ubiquitination site (green) that are enriched in D2-I3D suggest that the D2-I dimerization interface becomes more accessible in the phosphomimetic complex. These crosslinks are displayed on the surface representation of the D2-IWT model (PDB 6TNG), which is in an open state, and the DNA-bound D2-I3D model (from this study), which is in a closed state to show that these residues are buried in the dimerization surface in the open form, but become accessible in the closed form of the D2-I complex.

Supplementary information

Supplementary Information

Supplementary Table 1. Primers and oligonucleotides used in this study.

Reporting Summary

Peer Review File

Supplementary Video 1

Wild-type D2-I is in an open conformation in both the absence and presence of DNA. By contrast, phosphomimetic D2-I undergoes a large conformational rearrangement to stably close on dsDNA. This exposes the target lysines at the D2-I heterodimerization interface, promoting monoubiquitination by the FA core complex. Monoubiquitination locks the closed D2-I clamp on DNA, which then acts as a repair signal.

Supplementary Table 2

MS table including identified cross-links and accompanying data: cross-linked spectrum peptide match values, cross-link distances when displayed on the open or the closed D2-I form, and ratio comparison of abundance of quantified cross-links between the phosphomimetic and wild-type samples.

Source data

Source Data Fig. 1

Unprocessed SDS–PAGE gels and blots presented in Fig. 1.

Source Data Fig. 1

Numerical data used to generate the plot in Fig. 1c.

Source Data Fig. 4

Unprocessed EMSA gel scans presented in Fig. 4.

Source Data Fig. 4

Numerical data used to generate the plot in Fig. 4b.

Source Data Fig. 5

Unprocessed SDS–PAGE gels presented in Fig. 1.

Source Data Fig. 5

Numerical data used to generate the plot in Fig. 5d.

Source Data Fig. 6

Numerical data used to generate the plots in Fig. 6b and d.

Source Data Extended Data Fig. 1

Unprocessed SDS–PAGE gels presented in Extended Data Fig. 1.

Source Data Extended Data Fig. 4

Unprocessed SDS–PAGE gels presented in Extended Data Fig. 4.

Source Data Extended Data Fig. 4

Numerical data used to generate the plots in Extended Data Fig. 4a and c.

Source Data Extended Data Fig. 5

Unprocessed SDS–PAGE gels presented in Extended Data Fig. 5.

Source Data Extended Data Fig. 6

Unprocessed SDS–PAGE gels presented in Extended Data Fig. 6.

Source Data Extended Data Fig. 6

Numerical data used to generate the plot in Extended Data Fig. 6b.

Rights and permissions

Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sijacki, T., Alcón, P., Chen, Z.A. et al. The DNA-damage kinase ATR activates the FANCD2-FANCI clamp by priming it for ubiquitination. Nat Struct Mol Biol 29, 881–890 (2022). https://doi.org/10.1038/s41594-022-00820-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41594-022-00820-9

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing