Monoubiquitination by the human Fanconi Anemia core complex clamps FANCI:FANCD2 on DNA in filamentous arrays

  1. Winnie Tan
  2. Sylvie van Twest
  3. Andrew Leis
  4. Rohan Bythell-Douglas
  5. Vincent J Murphy
  6. Michael Sharp
  7. Michael W Parker
  8. Wayne M Crismani
  9. Andrew J Deans  Is a corresponding author
  1. St Vincent's Institute of Medical Research, Australia
  2. University of Melbourne, Australia

Abstract

FANCI:FANCD2 monoubiquitination is a critical event for replication fork stabilization by the Fanconi anemia (FA) DNA repair pathway. It has been proposed that at stalled replication forks, monoubiquitinated-FANCD2 serves to recruit DNA repair proteins that contain ubiquitin-binding motifs. Here we have reconstituted the FA pathway in vitro to study functional consequences of FANCI:FANCD2 monoubiquitination. We report that monoubiquitination does not promote any specific exogenous protein:protein interactions, but instead stabilizes FANCI:FANCD2 heterodimers on dsDNA. This clamping requires monoubiquitination of only the FANCD2 subunit. We further show that purified monoubiquitinated FANCI:FANCD2 forms filament-like arrays on long dsDNA using electron microscopy. Our results reveal how monoubiquitinated FANCI:FANCD2, defective in many cancer types and all cases of FA, is activated upon DNA binding.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Winnie Tan

    Genome Stability Unit, St Vincent's Institute of Medical Research, Fitzroy, Australia
    Competing interests
    The authors declare that no competing interests exist.
  2. Sylvie van Twest

    Genome Stability Unit, St Vincent's Institute of Medical Research, Fitzroy, Australia
    Competing interests
    The authors declare that no competing interests exist.
  3. Andrew Leis

    Department of Biochemistry, University of Melbourne, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  4. Rohan Bythell-Douglas

    Genome Stability Unit, St Vincent's Institute of Medical Research, Fitzroy, Australia
    Competing interests
    The authors declare that no competing interests exist.
  5. Vincent J Murphy

    Genome Stability Unit, St Vincent's Institute of Medical Research, Fitzroy, Australia
    Competing interests
    The authors declare that no competing interests exist.
  6. Michael Sharp

    Genome Stability Unit, St Vincent's Institute of Medical Research, Fitzroy, Australia
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1019-3729
  7. Michael W Parker

    Australian Cancer Research Foundation Rational Drug Discovery Centre, St Vincent's Institute of Medical Research, Fitzroy, Australia
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3101-1138
  8. Wayne M Crismani

    Genome Stability Unit, St Vincent's Institute of Medical Research, Fitzroy, Australia
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0143-8293
  9. Andrew J Deans

    Genome Stability Unit, St Vincent's Institute of Medical Research, Fitzroy, Australia
    For correspondence
    adeans@svi.edu.au
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5271-4422

Funding

National Health and Medical Research Council (GNT1123100 and GNT1181110)

  • Andrew J Deans

National Health and Medical Research Council (GNT1156343)

  • Wayne M Crismani

Fanconi Anemia Research Fund

  • Wayne M Crismani
  • Andrew J Deans

Victorian Government's OIS Program

  • Winnie Tan
  • Sylvie van Twest
  • Rohan Bythell-Douglas
  • Vincent J Murphy
  • Michael Sharp
  • Michael W Parker
  • Wayne M Crismani
  • Andrew J Deans

Maddie Riewoldt's Vision

  • Wayne M Crismani
  • Andrew J Deans

Victorian Cancer Agency

  • Andrew J Deans

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Wolf-Dietrich Heyer, University of California, Davis, United States

Publication history

  1. Received: December 3, 2019
  2. Accepted: March 12, 2020
  3. Accepted Manuscript published: March 13, 2020 (version 1)
  4. Version of Record published: April 14, 2020 (version 2)

Copyright

© 2020, Tan et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Winnie Tan
  2. Sylvie van Twest
  3. Andrew Leis
  4. Rohan Bythell-Douglas
  5. Vincent J Murphy
  6. Michael Sharp
  7. Michael W Parker
  8. Wayne M Crismani
  9. Andrew J Deans
(2020)
Monoubiquitination by the human Fanconi Anemia core complex clamps FANCI:FANCD2 on DNA in filamentous arrays
eLife 9:e54128.
https://doi.org/10.7554/eLife.54128

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