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ATM phosphorylation of Nijmegen breakage syndrome protein is required in a DNA damage response


Nijmegen breakage syndrome (NBS) is characterized by extreme radiation sensitivity, chromosomal instability and cancer1. The phenotypes are similar to those of ataxia telangiectasia mutated (ATM) disease, where there is a deficiency in a protein kinase that is activated by DNA damage, indicating that the Nbs and Atm proteins may participate in common pathways. Here we report that Nbs is specifically phosphorylated in response to γ-radiation, ultraviolet light and exposure to hydroxyurea. Phosphorylation of Nbs mediated by γ-radiation, but not that induced by hydroxyurea or ultraviolet light, was markedly reduced in ATM cells. In vivo, Nbs was phosphorylated on many serine residues, of which S343, S397 and S615 were phosphorylated by Atm in vitro. At least two of these sites were underphosphorylated in ATM cells. Inactivation of these serines by mutation partially abrogated Atm-dependent phosphorylation. Reconstituting NBS cells with a mutant form of Nbs that cannot be phosphorylated at selected, ATM-dependent serine residues led to a specific reduction in clonogenic survival after γ-radiation. Thus, phosphorylation of Nbs by Atm is critical for certain responses of human cells to DNA damage.

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Figure 1: DNA-damage-induced phosphorylation of Nbs.
Figure 2: The Atm kinase regulates phosphorylation of Nbs after γ-irradiation.
Figure 3: Analysis of possible sites of ATM phosphorylation of Nbs.
Figure 4: Failure of certain phosphosite mutants of Nbs to protect NBS fibroblasts from γ-irradiation-induced death.

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We thank P. Concannon, Y. Shiloh and R. Abraham for distribution of cell lines; J. Gan and J. DeCaprio for help with monoclonal antibody production; members of the Livingston and Weaver labs for discussions; and R. DePinho, D. Kufe, F. Alt, S. Kharbanda and M. Weber for comments and criticism and for reading the manuscript. This work was supported by funds from the NIH and the March of Dimes Foundation. X.W. was supported by a fellowship from the Jane Coffin Childs Memorial Fund for Medical Research.

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Wu, X., Ranganathan, V., Weisman, D. et al. ATM phosphorylation of Nijmegen breakage syndrome protein is required in a DNA damage response. Nature 405, 477–482 (2000).

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