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ATR and ATM regulate the timing of DNA replication origin firing

Nature Cell Biology volume 6, pages 648–655 (2004)Cite this article

Abstract

Timing of DNA replication initiation is dependent on S-phase-promoting kinase (SPK) activity at discrete origins and the simultaneous function of many replicons1,2. DNA damage prevents origin firing through the ATM- and ATR-dependent inhibition of Cdk2 and Cdc7 SPKs3,4. Here, we establish that modulation of ATM- and ATR-signalling pathways controls origin firing in the absence of DNA damage. Inhibition of ATM and ATR with caffeine or specific neutralizing antibodies, or upregulation of Cdk2 or Cdc7, promoted rapid and synchronous origin firing; conversely, inhibition of Cdc25A slowed DNA replication. Cdk2 was in equilibrium between active and inactive states, and the concentration of replication protein A (RPA)-bound single-stranded DNA (ssDNA) correlated with Chk1 activation and inhibition of origin firing. Furthermore, ATM was transiently activated during ongoing replication. We propose that ATR and ATM regulate SPK activity through a feedback mechanism originating at active replicons. Our observations establish that ATM- and ATR-signalling pathways operate during an unperturbed cell cycle to regulate initiation and progression of DNA synthesis, and are therefore poised to halt replication in the presence of DNA damage.

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Figure 1: Caffeine accelerates the initiation of replication.
Figure 2: The effect of caffeine on replication is the result of ATM and ATR inhibition.
Figure 3: Regulation of firing and replication by rate-limiting SPKs and Cdc25A.
Figure 4: ssDNA transiently accumulates and modulates origin firing.
Figure 5: ssDNA regulates the caffeine-sensitive inhibition of origin firing.

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Acknowledgements

We thank J. Walter for help and advice in setting up the NPE system; G.-H. Lee and T.T. Paull for the generous gift of purified ATM protein; K. Cimprich for the ATR antibody; J. Maller for Cdc25A baculovirus; M. Michael for the Geminin construct; D. Grieco for the p21 construct; H. Takisawa for the Cdc45 antibody; P. Jackson for RPA antibody; J. Boroweic for purified hRPA protein; E. Kim for production of recombinant Cdk2–Cyclin E and Cdc25A; and C. Ying for recombinant Cdc45. We also thank members of the Gautier lab for helpful discussions and critical reading of the manuscript. This work was supported by grants from the National Institutes of Health (RO1 CA92245) and the American Cancer Society (RSG CCG-103367) to J.G.

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  1. David Shechter

    Present address: The Laboratory of Chromatin Biology, Rockefeller University, 1230 York Avenue, New York, NY 10021, USA

Authors and Affiliations

  1. Integrated Program in Cellular, Molecular, and Biophysical Studies, Columbia University College of Physicians and Surgeons, 701 West 168th Street, New York, 10032, NY, USA

    David Shechter

  2. Department of Genetics and Development, Hammer Health Sciences Center Room 1620, Columbia University College of Physicians and Surgeons, 701 West 168th Street, New York, 10032, NY, USA

    David Shechter, Vincenzo Costanzo & Jean Gautier

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Correspondence to Jean Gautier.

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Shechter, D., Costanzo, V. & Gautier, J. ATR and ATM regulate the timing of DNA replication origin firing. Nat Cell Biol 6, 648–655 (2004). https://doi.org/10.1038/ncb1145

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