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53BP1-dependent robust localized KAP-1 phosphorylation is essential for heterochromatic DNA double-strand break repair


DNA double-strand breaks (DSBs) trigger ATM (ataxia telangiectasia mutated) signalling and elicit genomic rearrangements and chromosomal fragmentation if misrepaired or unrepaired. Although most DSB repair is ATM-independent, 15% of ionizing radiation (IR)-induced breaks persist in the absence of ATM-signalling1. 53BP1 (p53-binding protein 1) facilitates ATM-dependent DSB repair but is largely dispensable for ATM activation or checkpoint arrest. ATM promotes DSB repair within heterochromatin by phosphorylating KAP-1 (KRAB-associated protein 1, also known as TIF1β, TRIM28 or KRIP-1; ref. 2). Here, we show that the ATM signalling mediator proteins MDC1, RNF8, RNF168 and 53BP1 are also required for heterochromatic DSB repair. Although KAP-1 phosphorylation is critical for 53BP1-mediated repair, overall phosphorylated KAP-1 (pKAP-1) levels are only modestly affected by 53BP1 loss. pKAP-1 is transiently pan-nuclear but also forms foci overlapping with γH2AX in heterochromatin. Cells that do not form 53BP1 foci, including human RIDDLE (radiosensitivity, immunodeficiency, dysmorphic features and learning difficulties) syndrome cells, fail to form pKAP-1 foci. 53BP1 amplifies Mre11–NBS1 accumulation at late-repairing DSBs, concentrating active ATM and leading to robust, localized pKAP-1. We propose that ionizing-radiation induced foci (IRIF) spatially concentrate ATM activity to promote localized alterations in regions of chromatin otherwise inhibitory to repair.

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Figure 1: 53BP1, RNF8 and MDC1 are essential for heterochromatic DSB repair by promoting KAP-1 phosphorylation.
Figure 2: Discrete foci of pKAP-1 persist at late-repairing IR-induced foci.
Figure 3: Cells which fail to form 53BP1 foci fail to maintain high levels of pKAP-1 in the vicinity of a DSB.
Figure 4: 53BP1 concentrates active ATM at sites of damage.
Figure 5: 53BP1 promotes MRN hyper-accumulation at sites of damage.


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We thank T. Paull for discussions of unpublished data, A. Vindigni for mass spectrometry analysis, K. Iwabuchi for 53BP1 expression constructs and Y. Shiloh and Y. Ziv for providing KAP-1 expression constructs. A.A.G. was supported by grants from the Alberta Heritage Foundation for Medical Research and the Association for International Cancer Research (AICR). The P.A.J. laboratory is funded by the MRC, AICR, the Wellcome Research Fund, the Department of Health (UK) and the EU integrated projects on DNA-Repair (LSHG-CT-2005-512113) and Risc-Rad (FI6R-CT-2003-508842).

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All authors contributed towards scientific discussion and manuscript editing. A.T.N. and A.A.G. performed the experiments. N.R. and M.L. provided PFGE data and, with A.S., first characterized the 53BP1/MDC1 DSB repair defect. G.S.S. provided RIDDLE syndrome cells complemented ± RNF168. A.A.G. and P.A.J. co-authored the manuscript and conceived and designed the study.

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Correspondence to Penelope A. Jeggo or Aaron A. Goodarzi.

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The authors declare no competing financial interests.

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Noon, A., Shibata, A., Rief, N. et al. 53BP1-dependent robust localized KAP-1 phosphorylation is essential for heterochromatic DNA double-strand break repair. Nat Cell Biol 12, 177–184 (2010).

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