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ATM damage response and XLF repair factor are functionally redundant in joining DNA breaks

Abstract

Classical non-homologous DNA end-joining (NHEJ) is a major mammalian DNA double-strand-break (DSB) repair pathway. Deficiencies for classical NHEJ factors, such as XRCC4, abrogate lymphocyte development, owing to a strict requirement for classical NHEJ to join V(D)J recombination DSB intermediates1,2. The XRCC4-like factor (XLF; also called NHEJ1) is mutated in certain immunodeficient human patients and has been implicated in classical NHEJ3,4,5,6; however, XLF-deficient mice have relatively normal lymphocyte development and their lymphocytes support normal V(D)J recombination5. The ataxia telangiectasia-mutated protein (ATM) detects DSBs and activates DSB responses by phosphorylating substrates including histone H2AX7. However, ATM deficiency causes only modest V(D)J recombination and lymphocyte developmental defects, and H2AX deficiency does not have a measurable impact on these processes7,8,9. Here we show that XLF, ATM and H2AX all have fundamental roles in processing and joining DNA ends during V(D)J recombination, but that these roles have been masked by unanticipated functional redundancies. Thus, combined deficiency of ATM and XLF nearly blocks mouse lymphocyte development due to an inability to process and join chromosomal V(D)J recombination DSB intermediates. Combined XLF and ATM deficiency also severely impairs classical NHEJ, but not alternative end-joining, during IgH class switch recombination. Redundant ATM and XLF functions in classical NHEJ are mediated by ATM kinase activity and are not required for extra-chromosomal V(D)J recombination, indicating a role for chromatin-associated ATM substrates. Correspondingly, conditional H2AX inactivation in XLF-deficient pro-B lines leads to V(D)J recombination defects associated with marked degradation of unjoined V(D)J ends, revealing that H2AX has a role in this process.

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Figure 1: ATM and XLF have redundant functions in lymphocyte development.
Figure 2: ATM and XLF have redundant functions in chromosomal V(D)J recombination.
Figure 3: ATM and XLF synergize in classical NHEJ during CSR.
Figure 4: H2AX and XLF have redundant functions.

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Acknowledgements

We thank Y. Fujiwara and P. Huang for technical support. We thank B. Sleckman for advice, reagents and for critical review of this manuscript. This work is supported by NIH grant AI076210 and AI020047 to F.W.A. F.W.A. is an investigator of the Howard Hughes Medical Institute. S.Z. was a fellow, then senior fellow of Leukemia and Lymphomas Society of America and a St Baldrick Scholar. C.G. and Y.Z. are fellows of Cancer Research Institute. C.B. receives support from the pre-doctoral training program of Cancer Research Institute. D.R.W. is supported by a career development award from AAAI/GlaxoSmithKline and by NIH training grant AI007376.

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S.Z., C.G. and F.W.A. designed experiments and wrote the paper. S.Z., C.G., C.B., V.O., H.-L.C., Y.Z., D.R.W., G.Y., H.P., P.H.G. and R.L.D. performed experiments.

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Correspondence to Frederick W. Alt.

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Zha, S., Guo, C., Boboila, C. et al. ATM damage response and XLF repair factor are functionally redundant in joining DNA breaks. Nature 469, 250–254 (2011). https://doi.org/10.1038/nature09604

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