Abstract
DNA double-strand breaks (DSBs) are generated by the recombination activating gene (RAG) endonuclease in all developing lymphocytes as they assemble antigen receptor genes1. DNA cleavage by RAG occurs only at the G1 phase of the cell cycle and generates two hairpin-sealed DNA (coding) ends that require nucleolytic opening before their repair by classical non-homologous end-joining (NHEJ)1,2,3. Although there are several cellular nucleases that could perform this function, only the Artemis nuclease is able to do so efficiently2,3. Here, in vivo, we show that in murine cells the histone protein H2AX prevents nucleases other than Artemis from processing hairpin-sealed coding ends; in the absence of H2AX, CtIP can efficiently promote the hairpin opening and resection of DNA ends generated by RAG cleavage. This CtIP-mediated resection is inhibited by γ-H2AX and by MDC-1 (mediator of DNA damage checkpoint 1), which binds to γ-H2AX in chromatin flanking DNA DSBs. Moreover, the ataxia telangiectasia mutated (ATM) kinase activates antagonistic pathways that modulate this resection. CtIP DNA end resection activity is normally limited to cells at post-replicative stages of the cell cycle, in which it is essential for homology-mediated repair4,5. In G1-phase lymphocytes, DNA ends that are processed by CtIP are not efficiently joined by classical NHEJ and the joints that do form frequently use micro-homologies and show significant chromosomal deletions. Thus, H2AX preserves the structural integrity of broken DNA ends in G1-phase lymphocytes, thereby preventing these DNA ends from accessing repair pathways that promote genomic instability.
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Acknowledgements
We thank E. Oltz and F. Alt for critical review of the manuscript; R. Baer for providing us with the anti-CtIP antibodies; and G. Longmore and Y. Feng for pFLRU, pHR′Δ8.2R and pCMV-VSVg. This work is supported by National Institutes of Health (NIH) grants AI074953 (B.P.S.), AI47829 (B.P.S.), CA136470 (B.P.S. and C.H.B.), CA125195 (C.H.B.), CA21765 (P.J.McK.) and NS37956 (P.J.McK.). J.J.B. is supported by a NIH Ruth L. Kirschstein National Research Service Award (T32 HD007499) and a Children’s Discovery Institute Fellows Award. C.H.B. was a Pew Scholar in the Biomedical Sciences program and is a Leukemia and Lymphoma Society Scholar.
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B.P.S. and B.A.H. conceived the study and wrote the paper. B.P.S. and C.H.B. designed experiments and interpreted data. B.A.H., A.T.T., Y.D., G.S., J.Z. and J.J.B. designed and performed experiments and interpreted data. Z.F. and L.M.W. performed experiments. P.J.McK. provided important reagents.
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Helmink, B., Tubbs, A., Dorsett, Y. et al. H2AX prevents CtIP-mediated DNA end resection and aberrant repair in G1-phase lymphocytes. Nature 469, 245–249 (2011). https://doi.org/10.1038/nature09585
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DOI: https://doi.org/10.1038/nature09585
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