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Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks

Nature Cell Biology volume 5, pages 675–679 (2003)Cite this article

Abstract

Histone H2AX is rapidly phosphorylated in the chromatin micro-environment surrounding a DNA double-strand break (DSB). Although H2AX deficiency is not detrimental to life, H2AX is required for the accumulation of numerous essential proteins into irradiation induced foci (IRIF). However, the relationship between IRIF formation, H2AX phosphorylation (γ-H2AX) and the detection of DNA damage is unclear. Here, we show that the migration of repair and signalling proteins to DSBs is not abrogated in H2AX−/− cells, or in H2AX-deficient cells that have been reconstituted with H2AX mutants that eliminate phosphorylation. Despite their initial recruitment to DSBs, numerous factors, including Nbs1, 53BP1 and Brca1, subsequently fail to form IRIF. We propose that γ-H2AX does not constitute the primary signal required for the redistribution of repair complexes to damaged chromatin, but may function to concentrate proteins in the vicinity of DNA lesions. The differential requirements for factor recruitment to DSBs and sequestration into IRIF may explain why essential regulatory pathways controlling the ability of cells to respond to DNA damage are not abolished in the absence of H2AX.

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Figure 1: H2AX-independent recruitment of Nbs1, 53BP1 and Brca1 to DSBs.
Figure 2: Time-dependent changes in 53BP1 staining at DSBs.
Figure 3: γ-H2AX and ATM are not required for M–R–N recruitment to DSBs.

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Acknowledgements

We thank M. Gellert, A. Singer and R. Hodes for critical comments on the manuscript. We also thank D. Sackett for assistance, Y. Pommier and P. Leder for kindly providing the ATM-deficient cells, S. Ganesan and J. Chen for kindly providing Brca1 and 53BP1 antibodies. In addition, we also thank R. Schroff and M. Lichten for sharing unpublished data.

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Author notes

  1. Arkady Celeste and Oscar Fernandez-Capetillo: These authors contributed equally to this work.

Authors and Affiliations

  1. Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, 20892, Maryland, USA

    Arkady Celeste, Oscar Fernandez-Capetillo, Michael J. Kruhlak, David W. Staudt, Alicia Lee & André Nussenzweig

  2. Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, 20892, Maryland, USA

    Duane R. Pilch & William M. Bonner

  3. Section on Medical Biophysics, Laboratory of Integrative and Medical Biophysics, National Institutes of Health, Bethesda, 20892, Maryland, USA

    Robert F. Bonner

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  1. Arkady Celeste
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Correspondence to André Nussenzweig.

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Supplementary information

Figure S1. IR-induced redistribution of Nbs1 from PCNA clusters is H2AX independent.

Figure S2. Typing of reconstituted P53-/-H2AX-/- cells by 2-D gel analysis of histones. (PDF 1297 kb)

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Celeste, A., Fernandez-Capetillo, O., Kruhlak, M. et al. Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks. Nat Cell Biol 5, 675–679 (2003). https://doi.org/10.1038/ncb1004

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