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Rag mutations reveal robust alternative end joining

Abstract

Mammalian cells repair DNA double-strand breaks (DSBs) through either homologous recombination or non-homologous end joining (NHEJ). V(D)J recombination, a cut-and-paste mechanism for generating diversity in antigen receptors, relies on NHEJ for repairing DSBs introduced by the Rag1–Rag2 protein complex. Animals lacking any of the seven known NHEJ factors are therefore immunodeficient1. Nevertheless, DSB repair is not eliminated entirely in these animals: evidence of a third mechanism, ‘alternative NHEJ’, appears in the form of extremely rare V(D)J junctions2,3,4 and a higher rate of chromosomal translocations5,6. The paucity of these V(D)J events has suggested that alternative NHEJ contributes little to a cell’s overall repair capacity, being operative only (and inefficiently) when classical NHEJ fails. Here we find that removing certain portions of murine Rag proteins reveals robust alternative NHEJ activity in NHEJ-deficient cells and some alternative joining activity even in wild-type cells. We propose a two-tier model in which the Rag proteins collaborate with NHEJ factors to preserve genomic integrity during V(D)J recombination.

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Figure 1: FS361 and Stop361 Rag2 mutants allow use of homologous recombination and alternative NHEJ in wild-type cells.
Figure 2: Rag2 truncation mutants bypass genetic defects in classical NHEJ (DNA-PKcs).
Figure 3: Rag truncation mutants bypass genetic defects in classical NHEJ (Xrcc4).
Figure 4: FS361 and Stop361 bypass Xrcc4 deficiency using integrated substrates.

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Acknowledgements

This work was supported by NIH grants (to K.M. and D.B.R.) and the Irene Diamond Fund (D.B.R.). L.D. is a Fellow of The Leukemia and Lymphoma Society and G.R.W. is a Cancer Research Institute Postdoctoral Fellow. We thank J. Qiu for help with the mutant library and S. Salus for help during the early phases of this work.

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Correspondence to David B. Roth.

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Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-8 with Legends and Supplementary Tables 1-3. The Supplementary Figure 1 shows a two-tier model for preserving genomic integrity during V(D)J recombination. The Supplementary Figures 2-8 provide schematics of the assays, control data, and raw data on which the graphs in the figures are based. The Supplementary Tables 1 provide some sequences and analyses of junctions formed by the mutant Rag2 proteins in NHEJ-deficient cells. (PDF 839 kb)

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Corneo, B., Wendland, R., Deriano, L. et al. Rag mutations reveal robust alternative end joining. Nature 449, 483–486 (2007). https://doi.org/10.1038/nature06168

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