Abstract

Activation of the ATR kinase following perturbations to DNA replication relies on a complex mechanism involving ATR recruitment to RPA-coated single-stranded DNA via its binding partner ATRIP and stimulation of ATR kinase activity by TopBP1. Here, we discovered an independent ATR activation pathway in vertebrates, mediated by the uncharacterized protein ETAA1 (Ewing’s tumour-associated antigen 1). Human ETAA1 accumulates at DNA damage sites via dual RPA-binding motifs and promotes replication fork progression and integrity, ATR signalling and cell survival after genotoxic insults. Mechanistically, this requires a conserved domain in ETAA1 that potently and directly stimulates ATR kinase activity independently of TopBP1. Simultaneous loss of ETAA1 and TopBP1 gives rise to synthetic lethality characterized by massive genome instability and abrogation of ATR-dependent signalling. Our findings demonstrate that parallel TopBP1- and ETAA1-mediated pathways underlie ATR activation and that their combined action is essential for coping with replication stress.

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Acknowledgements

We thank D. Cortez and A. Kumagai for providing reagents. We thank K. Mayr, I. Paron and G. Sowa for mass spectrometry support and M. Steger and B. Splettstösser (all from Max Planck Institute of Biochemistry) for experimental advice. This work was supported by grants from The Novo Nordisk Foundation (grants no. NNF14CC0001 and NNF12OC0002114), European Research Council (ERC, grant agreement no. 616236 (DDRegulation)), The Danish Cancer Society, The Danish Council for Independent Research, Danish National Research Foundation (grant no. DNRF115) and Center for Integrated Protein Science Munich (CIPSM).

Author information

Author notes

    • Saskia Hoffmann
    • , Maxim A. X. Tollenaere
    •  & Teresa Ho

    These authors contributed equally to this work.

    • Markus Räschle

    Present address: Department of Molecular Genetics, TU Kaiserslautern, Paul-Ehrlich Strasse 24, 67663 Kaiserslautern, Germany.

Affiliations

  1. The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark

    • Peter Haahr
    • , Saskia Hoffmann
    • , Maxim A. X. Tollenaere
    • , Teresa Ho
    • , Luis Ignacio Toledo
    • , Simon Bekker-Jensen
    •  & Niels Mailand
  2. Center for Chromosome Stability, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark

    • Teresa Ho
    •  & Niels Mailand
  3. Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany

    • Matthias Mann
    •  & Markus Räschle

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Contributions

P.H. and N.M. conceived the study. P.H., S.H., M.A.X.T. and T.H. designed and performed cell-, biochemistry- and imaging-based experiments and analysed the data, under the supervision of N.M. M.R. designed and performed mass spectrometry experiments and analysed the data, under the supervision of M.M. L.I.T. provided help and support with quantitative image analysis and time-lapse microscopy. S.B.-J. co-supervised the study. N.M. wrote the manuscript with inputs from P.H. and M.R. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Markus Räschle or Niels Mailand.

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Videos

  1. 1.

    Representative example of mitotic progression in cells lacking TopBP1.

    HCT116 WT cells stably reconstituted with GFP-H2B were transfected with TopBP1 siRNA and monitored by live cell fluorescence microscopy at 24–60 h after siRNA transfection. Video shows representative examples of mitosis under these conditions.

  2. 2.

    Representative example of mitotic progression in cells lacking ETAA1 and TopBP1.

    HCT116 ETAA1Δ-3 cells stably reconstituted with GFP-H2B were transfected with TopBP1 siRNA and followed by live cell fluorescence microscopy at 24–60  h after siRNA transfection. Video shows representative examples of mitotic errors leading to nuclear fragmentation under these conditions.

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DOI

https://doi.org/10.1038/ncb3422

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