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CtIP promotes microhomology-mediated alternative end joining during class-switch recombination

Abstract

Immunoglobulin heavy chain (Igh locus) class-switch recombination (CSR) requires targeted introduction of DNA double strand breaks (DSBs) into repetitive 'switch'-region DNA elements in the Igh locus and subsequent ligation between distal DSBs. Both canonical nonhomologous end joining (C-NHEJ) that seals DNA ends with little or no homology and a poorly defined alternative end joining (A-NHEJ, also known as alt-NHEJ) process that requires microhomology ends for ligation have been implicated in CSR. Here, we show that the DNA end-processing factor CtIP is required for microhomology-directed A-NHEJ during CSR. Additionally, we demonstrate that microhomology joins that are enriched upon depletion of the C-NHEJ component Ku70 require CtIP. Finally, we show that CtIP binds to switch-region DNA in a fashion dependent on activation-induced cytidine deaminase. Our results establish CtIP as a bona fide component of microhomology-dependent A-NHEJ and unmask a hitherto unrecognized physiological role of microhomology-mediated end joining in a C-NHEJ–proficient environment.

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Figure 1: CtIP knockdown alters CSR in CH12 cells.
Figure 2: CtIP knockdown alters end joining during CSR.
Figure 3: CtIP knockdown alters end joining in Ku70-deficient cells.
Figure 4: CtIP binds to S-region DNA.
Figure 5: A model for the role of CtIP in CSR.

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Acknowledgements

We thank M. Jasin and colleagues for sharing unpublished results and members of the Chaudhuri laboratory for discussion and advice. This work was supported by grants from the Damon Runyon Cancer Research Fund, Alfred Bressler Foundation, Sloan-Kettering Institute and US National Institutes of Health (1R01AI072194-01A2).

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Contributions

M.L.-T., A.J.M. and D.K. conducted experiments; A.J.M. and S.Z. devised protocols for shRNA-mediated knockdown in CH12 cell lines; M.L.-T. and J.C. devised experiments, analyzed data and wrote the manuscript.

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Correspondence to Jayanta Chaudhuri.

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The authors declare no competing financial interests.

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Supplementary Figures 1–6 and Supplementary Tables 1–2 (PDF 1010 kb)

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Lee-Theilen, M., Matthews, A., Kelly, D. et al. CtIP promotes microhomology-mediated alternative end joining during class-switch recombination. Nat Struct Mol Biol 18, 75–79 (2011). https://doi.org/10.1038/nsmb.1942

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