Abstract
Class switch recombination (CSR) is a region-specific DNA recombination reaction that replaces one immunoglobulin heavy-chain constant region (Ch) gene with another. This enables a single variable (V) region gene to be used in conjunction with different downstream Ch genes, each having a unique biological activity. The molecular mechanisms that mediate CSR have not been defined, but activation-induced cytidine deaminase (AID), a putative RNA-editing enzyme, is required for this reaction1. Here we report that the Nijmegen breakage syndrome protein (Nbs1) and phosphorylated H2A histone family member X (γ-H2AX, also known as γ-H2afx), which facilitate DNA double-strand break (DSB) repair2,3,4, form nuclear foci at the Ch region in the G1 phase of the cell cycle in cells undergoing CSR, and that switching is impaired in H2AX-/- mice. Localization of Nbs1 and γ-H2AX to the Igh locus during CSR is dependent on AID. In addition, AID is required for induction of switch region (Sµ)-specific DNA lesions that precede CSR. These results place AID function upstream of the DNA modifications that initiate CSR.
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Acknowledgements
We thank F. Roschenthaler for Igκ probes; S. Sharrow for help with FACS; S. Ganesan for Brca1 antibodies; and A. Singer, E. Max, M. Gellert, R. Hodes and E. Besmer for comments on the manuscript and discussions. This work was supported in part by grants from the National Institutes of Health and the Leukemia Society to M.C.N. M.C.N. is a Howard Hughes Medical Institute investigator. The first two authors (S.P. and R.C.) contributed equally to this work.
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Figure 1. Inactivation of mouse H2AX.
(JPG 18.97 KB)
a, Genomic structure of the wild-type H2AX locus and targeting vector. A genomic clone covering the H2AX locus was isolated from a 129/Sv mouse lambda phage library (Stratagene). The targeting construct was designed as a null mutation that replaces a segment of H2AX between the unique Asc1 and Xho1 site in the coding sequence by a neomycin resistance cassette. b, Southern blot of XhoI-digested tail DNA from wildtype (+/+), heterozygous (+/-) and homozygous (-/-) H2AX-targeted mice hybridized with a probe indicated in a. The wild-type and mutant fragments are 8.8 and 10.6 kb respectively.
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Petersen, S., Casellas, R., Reina-San-Martin, B. et al. AID is required to initiate Nbs1/γ-H2AX focus formation and mutations at sites of class switching. Nature 414, 660–665 (2001). https://doi.org/10.1038/414660a
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DOI: https://doi.org/10.1038/414660a
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