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Nat Commun. 2017 Jun 12;8:15760. doi: 10.1038/ncomms15760.

Identification of the elementary structural units of the DNA damage response.

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Department of Biology, Technische Universität Darmstadt, 64287 Darmstadt, Germany.
Department of Biology II, Center for Integrated Protein Science Munich (CIPSM), LMU Munich, 82152 Planegg-Martinsried, Germany.
Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.
Department of Biophysics, GSI Helmholtzzentrum für Schwerionenforschung, 64291 Darmstadt, Germany.


Histone H2AX phosphorylation is an early signalling event triggered by DNA double-strand breaks (DSBs). To elucidate the elementary units of phospho-H2AX-labelled chromatin, we integrate super-resolution microscopy of phospho-H2AX during DNA repair in human cells with genome-wide sequencing analyses. Here we identify phospho-H2AX chromatin domains in the nanometre range with median length of ∼75 kb. Correlation analysis with over 60 genomic features shows a time-dependent euchromatin-to-heterochromatin repair trend. After X-ray or CRISPR-Cas9-mediated DSBs, phospho-H2AX-labelled heterochromatin exhibits DNA decondensation while retaining heterochromatic histone marks, indicating that chromatin structural and molecular determinants are uncoupled during repair. The phospho-H2AX nano-domains arrange into higher-order clustered structures of discontinuously phosphorylated chromatin, flanked by CTCF. CTCF knockdown impairs spreading of the phosphorylation throughout the 3D-looped nano-domains. Co-staining of phospho-H2AX with phospho-Ku70 and TUNEL reveals that clusters rather than nano-foci represent single DSBs. Hence, each chromatin loop is a nano-focus, whose clusters correspond to previously known phospho-H2AX foci.

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