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MDC1 is required for the intra-S-phase DNA damage checkpoint

Abstract

MRE11, RAD50 and NBS1 form a highly conserved protein complex (the MRE11 complex) that is involved in the detection, signalling and repair of DNA damage1. We identify MDC1 (KIAA0170/NFBD1), a protein that contains a forkhead-associated (FHA) domain and two BRCA1 carboxy-terminal (BRCT) domains, as a binding partner for the MRE11 complex. We show that, in response to ionizing radiation, MDC1 is hyperphosphorylated in an ATM-dependent manner, and rapidly relocalizes to nuclear foci that also contain the MRE11 complex, phosphorylated histone H2AX and 53BP1. Downregulation of MDC1 expression by small interfering RNA yields a radio-resistant DNA synthesis (RDS) phenotype and prevents ionizing radiation-induced focus formation by the MRE11 complex. However, downregulation of MDC1 does not abolish the ionizing radiation-induced phosphorylation of NBS1, CHK2 and SMC1, or the degradation of CDC25A. Furthermore, we show that overexpression of the MDC1 FHA domain interferes with focus formation by MDC1 itself and by the MRE11 complex, and induces an RDS phenotype. These findings reveal that MDC1-mediated focus formation by the MRE11 complex at sites of DNA damage is crucial for the efficient activation of the intra-S-phase checkpoint.

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Figure 1: Association of MDC1 with the MRE11 complex.
Figure 2: MDC1 responds to ionizing radiation.
Figure 3: MDC1 focus formation does not require ATM, ATR, DNA-PK or NBS1.
Figure 5: MDC1 functions in the S-phase checkpoint.
Figure 4: MDC1 is required to recruit the MRE11 complex to foci.

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Acknowledgements

We thank Y. Shiloh for AT complemented cells, T. D. Halazonetis for anti-53BP1 antibodies, J. Petrini for anti-MRE11 and anti-NBS1 antibodies, and M. Kastan and K. K. Khanna for anti-NBS1-S343(P) antibodies. We thank S.P.J. laboratory members for helpful discussions and critical reading of the manuscript, especially J. Bradbury, F. d'Adda di Fagagna, M. Grenon, J. Rouse and V. Smits. We thank S. Elledge for sharing unpublished results. This study was supported by grants from the Association for International Cancer Research, the A-T Medical Research Trust and the Swiss National Foundation. Research in the S.P.J. laboratory is funded by Cancer Research UK.

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Correspondence to Stephen P. Jackson.

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S. Jackson founded KuDOS Pharmaceuticals Ltd and is chief scientific officer of the company. KuDOS is using knowledge of DNA repair pathways to discover and develop new therapies for cancer and other human diseases.

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Goldberg, M., Stucki, M., Falck, J. et al. MDC1 is required for the intra-S-phase DNA damage checkpoint. Nature 421, 952–956 (2003). https://doi.org/10.1038/nature01445

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