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Distinct spatiotemporal dynamics of mammalian checkpoint regulators induced by DNA damage

Nature Cell Biology volume 5, pages 255–260 (2003)Cite this article

Abstract

Cell cycle checkpoints are signal transduction pathways activated after DNA damage to protect genomic integrity1. Dynamic spatiotemporal coordination is a vital, but poorly understood aspect, of these checkpoints. Here, we provide evidence for a strikingly different behaviour of Chk2 versus Nbs1, key mediators of the ataxia-telangiecatesia-mutated (ATM)-controlled checkpoint pathways induced by DNA double-strand breaks (DSBs)1,2. In live human cells with DSBs restricted to small sub-nuclear areas, Nbs1 was rapidly recruited to the damaged regions and underwent a dynamic exchange in the close vicinity of the DSB sites. In contrast, Chk2 continued to rapidly move throughout the entire nucleus, irrespective of DNA damage and including the DSB-free areas. Although phosphorylation of Chk2 by ATM occurred exclusively at the DSB sites, forced immobilization of Chk2 to spatially restricted, DSB-containing nuclear areas impaired its stimulating effect on p53-dependent transcription. These results unravel a dynamic nature of Nbs1 interaction with DSB lesions and identify Chk2 as a candidate transmitter of the checkpoint signal, allowing for a coordinated pan-nuclear response to focal DNA damage.

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Figure 1: Sub-nuclear-restricted DSBs elicit a pan-nuclear Chk2 response.
Figure 2: ATM-phosphorylated Nbs1 accumulates exclusively on the sites of DSBs.
Figure 3: Rapid movement of the Chk2 protein in vivo.
Figure 4: Dynamic interaction of Nbs1 with the sites of DSB in vivo.

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Acknowledgements

We thank J. Chen, T. Halazonetis, M. Kastan, J. Petrini, M. Oren and G. Wahl for providing reagents, and C. Lindeneg, P. Geltzer and R. Kern for their excellent technical assistance. We are grateful to the Danish Cancer Society, European Commission, and the John and Birthe Meyer Foundation for financial support.

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  1. Claudia Lukas and Jacob Falck: These authors contributed equally to this work.

Authors and Affiliations

  1. Danish Cancer Society, Institute of Cancer Biology, Strandboulevarden 49, Copenhagen, DK-2100, Denmark

    Claudia Lukas, Jacob Falck, Jirina Bartkova, Jiri Bartek & Jiri Lukas

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Correspondence to Jiri Lukas.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

Fig. S1 Generation of DSB restricted to defined subnuclear regions. (PDF 1561 kb)

Fig. S2 Specificity determination of the phospho-Chk2-Thr68 antibodies.Fig. S2 Specificity determination of the phospho-Chk2-Thr68 antibodies.

Fig. S3 Pan-nuclear distribution of ATM-phosphorylated Chk2 and recruitment of the ATM-phosphorylated Nbs1 to the DSB sites confirmed by coimmmunostaining with phospho-histone H2AX.

Fig. S4 Specificity determination of the phospho-Nbs1-Ser343 antibody.

Fig. S5 GFP attachment does not impair the function of Chk2 and Nbs1, respectively.

Fig. S6 A strategy to immobilize Chk2 in vivo.

Fig. S7 Lack of specific recruitment of p53 to the DSB-containing nuclear areas.

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Lukas, C., Falck, J., Bartkova, J. et al. Distinct spatiotemporal dynamics of mammalian checkpoint regulators induced by DNA damage. Nat Cell Biol 5, 255–260 (2003). https://doi.org/10.1038/ncb945

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