Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

AID is required to initiate Nbs1/γ-H2AX focus formation and mutations at sites of class switching

Nature volume 414, pages 660–665 (2001)Cite this article

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: DNA repair foci in wild-type B lymphocytes after stimulation with LPS and IL-4 for 72 h.
Figure 2: DNA repair foci associated with CSR form predominantly in the G1 phase of the cell cycle.
Figure 3: Kinetics of CH-associated foci accumulation in wild-type mice and impaired switching in H2AX-/- mice.
Figure 4: Co-localization of Nbs1 and γ-H2AX at the Igh locus is dependent on AID.
Figure 5: AID-dependent Sµ mutation induced by LPS and IL-4.

Similar content being viewed by others

References

  1. Muramatsu, M. et al. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102, 553–563 (2000).

    Article  CAS  Google Scholar 

  2. Carney, J. P. et al. The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell 93, 477–486 (1998).

    Article  CAS  Google Scholar 

  3. Rogakou, E. P., Boon, C., Redon, C. & Bonner, W. M. Megabase chromatin domains involved in DNA double-strand breaks in vivo. J. Cell. Biol. 146, 905–916 (1999).

    Article  CAS  Google Scholar 

  4. Downs, J. A., Lowndes, N. F. & Jackson, S. P. A role for Saccharomyces cerevisiae histone H2A in DNA repair. Nature 408, 1001–1004 (2000).

    Article  ADS  CAS  Google Scholar 

  5. Revy, P. et al. Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the Hyper-IgM syndrome (HIGM2). Cell 102, 565–575 (2000).

    Article  CAS  Google Scholar 

  6. Paull, T. T. et al. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr. Biol. 10, 886–895 (2000).

    Article  CAS  Google Scholar 

  7. Li, M. J. et al. Rad51 expression and localization in B cells carrying out class switch recombination. Proc. Natl Acad. Sci. USA 93, 10222–10227 (1996).

    Article  ADS  CAS  Google Scholar 

  8. Scully, R. et al. Dynamic changes of BRCA1 subnuclear location and phosphorylation state are initiated by DNA damage. Cell 90, 425–435 (1997).

    Article  CAS  Google Scholar 

  9. Zhu, X. D., Kuster, B., Mann, M., Petrini, J. H. & Lange, T. Cell-cycle-regulated association of RAD50/MRE11/NBS1 with TRF2 and human telomeres. Nature Genet. 25, 347–352 (2000).

    Article  CAS  Google Scholar 

  10. Chen, H. T. et al. Response to RAG-mediated V(D)J Cleavage by NBS1 and γ-H2AX. Science 290, 1962–1964 (2000).

    Article  ADS  CAS  Google Scholar 

  11. Papavasiliou, F. N. & Schatz, D. G. Cell-cycle-regulated DNA double-stranded breaks in somatic hypermutation of immunoglobulin genes. Nature 408, 216–221 (2000).

    Article  ADS  CAS  Google Scholar 

  12. Sale, J. E. & Neuberger, M. S. TdT-accessible breaks are scattered over the immunoglobulin V domain in a constitutively hypermutating B cell line. Immunity 9, 859–869 (1998).

    Article  CAS  Google Scholar 

  13. Bross, L. et al. DNA double-strand breaks in immunoglobulin genes undergoing somatic hypermutation. Immunity 13, 589–597 (2000).

    Article  CAS  Google Scholar 

  14. Casellas, R. et al. Ku80 is required for immunoglobulin isotype switching. EMBO J. 17, 2404–2411 (1998).

    Article  CAS  Google Scholar 

  15. Manis, J. P. et al. Ku70 is required for late B cell development and immunoglobulin heavy chain class switching. J. Exp. Med. 187, 2081–2089 (1998).

    Article  CAS  Google Scholar 

  16. Rolink, A., Melchers, F. & Andersson, J. The SCID but not the RAG-2 gene product is required for Sµ-Sε heavy chain class switching. Immunity 5, 319–330 (1996).

    Article  CAS  Google Scholar 

  17. Stavnezer-Nordgren, J. & Sirlin, S. Specificity of immunoglobulin heavy chain switch correlates with activity of germline heavy chain genes prior to switching. EMBO J. 5, 95–102 (1986).

    Article  CAS  Google Scholar 

  18. Yancopoulos, G. D. et al. Secondary genomic rearrangement events in pre-B cells: VHDJH replacement by a LINE-1 sequence and directed class switching. EMBO J. 5, 3259–3266 (1986).

    Article  CAS  Google Scholar 

  19. Barnes, W. M. PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc. Natl Acad. Sci. USA 91, 2216–2220 (1994).

    Article  ADS  CAS  Google Scholar 

  20. Dunnick, W., Hertz, G. Z., Scappino, L. & Gritzmacher, C. DNA sequences at immunoglobulin switch region recombination sites. Nucleic Acids Res. 21, 365–372 (1993).

    Article  CAS  Google Scholar 

  21. Kinoshita, K. & Honjo, T. Linking class-switch recombination with somatic hypermutation. Nature Rev. Mol. Cell Biol. 2, 493–503 (2001).

    Article  CAS  Google Scholar 

  22. Winter, E., Krawinkel, U. & Radbruch, A. Directed Ig class switch recombination in activated murine B cells. EMBO J. 6, 1663–1671 (1987).

    Article  CAS  Google Scholar 

  23. Rogozin, I. B. & Kolchanov, N. A. Somatic hypermutagenesis in immunoglobulin genes. II. Influence of neighbouring base sequences on mutagenesis. Biochim. Biophys. Acta 1171, 11–18 (1992).

    Article  CAS  Google Scholar 

  24. Pasqualucci, L. et al. BCL-6 mutations in normal germinal center B cells: evidence of somatic hypermutation acting outside Ig loci. Proc. Natl Acad. Sci. USA 95, 11816–11821 (1998).

    Article  ADS  CAS  Google Scholar 

  25. Shen, H. M., Peters, A., Baron, B., Zhu, X. & Storb, U. Mutation of BCL-6 gene in normal B cells by the process of somatic hypermutation of Ig genes. Science 280, 1750–1752 (1998).

    Article  ADS  CAS  Google Scholar 

  26. Dunnick, W., Wilson, M. & Stavnezer, J. Mutations, duplication, and deletion of recombined switch regions suggest a role for DNA replication in the immunoglobulin heavy-chain switch. Mol. Cell. Biol. 9, 1850–1856 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Bemark, M. et al. Somatic hypermutation in the absence of DNA-dependent protein kinase catalytic subunit (DNA-PK(cs)) or recombination-activating gene (RAG)1 activity. J. Exp. Med. 192, 1509–1514 (2000).

    Article  CAS  Google Scholar 

  28. Sale, J. E., Calandrini, D. M., Takata, M., Takeda, S. & Neuberger, M. S. Ablation of XRCC2/3 transforms immunoglobulin V gene conversion into somatic hypermutation. Nature 412, 921–926 (2001).

    Article  ADS  CAS  Google Scholar 

  29. Brenner, S. & Milstein, C. Origin of antibody variation. Nature 211, 242–243 (1966).

    Article  ADS  CAS  Google Scholar 

  30. Casellas, R. et al. Contribution of receptor editing to the antibody repertoire. Science 291, 1541–1544 (2001).

    Article  ADS  CAS  Google Scholar 

Download references

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.

Author information

Author notes

  1. Duane R. Pilch, Christophe Redon, William M. Bonner, Michel C. Nussenzweig and André Nussenzweig: These authors contributed equally to this work

Authors and Affiliations

  1. Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, 20892, Maryland, USA

    Simone Petersen, Hua Tang Chen, Arkady Celeste & André Nussenzweig

  2. Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, 20892, Maryland, USA

    Michael J. Difilippantonio & Thomas Ried

  3. Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, 20892, Maryland, USA

    Duane R. Pilch, Christophe Redon & William M. Bonner

  4. Laboratory of Molecular Immunology, The Rockefeller University, and Howard Hughes Medical Institute, New York, 10021, New York, USA

    Rafael Casellas, Bernardo Reina-San-Martin, Patrick C. Wilson, Leif Hanitsch & Michel C. Nussenzweig

  5. Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501, Japan

    Masamichi Muramatsu & Tasuku Honjo

Authors
  1. Simone Petersen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rafael Casellas
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bernardo Reina-San-Martin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hua Tang Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael J. Difilippantonio
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Patrick C. Wilson
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Leif Hanitsch
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Arkady Celeste
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Masamichi Muramatsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Duane R. Pilch
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Christophe Redon
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Thomas Ried
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. William M. Bonner
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Tasuku Honjo
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Michel C. Nussenzweig
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. André Nussenzweig
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to André Nussenzweig.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

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.

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/414660a

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing