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.

  • Article
  • Published:

Multiple functions of MRN in end-joining pathways during isotype class switching

Nature Structural & Molecular Biology volume 16, pages 808–813 (2009)Cite this article

Abstract

The Mre11–Rad50–NBS1 (MRN) complex has many roles in response to DNA double-strand breaks, but its functions in repair by nonhomologous end joining (NHEJ) pathways are poorly understood. We have investigated requirements for MRN in class switch recombination (CSR), a programmed DNA rearrangement in B lymphocytes that requires NHEJ. To this end, we have engineered mice that lack the entire MRN complex in B lymphocytes or that possess an intact complex that harbors mutant Mre11 lacking DNA nuclease activities. MRN deficiency confers a strong defect in CSR, affecting both the classic and the alternative NHEJ pathways. In contrast, absence of Mre11 nuclease activities causes a milder phenotype, revealing a separation of function within the complex. We propose a model in which MRN stabilizes distant breaks and processes DNA termini to facilitate repair by both the classical and alternative NHEJ pathways.

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: Mre11 deficiencies in the B lymphocyte lineage.
Figure 2: Distinct deficiencies in isotype switching conferred by MRN and Atm mutations.
Figure 3: Requirement for MRN in the repair process.
Figure 4: Independence of γ-H2AX-Mdc1-53bp1 and MRN.

Similar content being viewed by others

References

  1. McKinnon, P.J. & Caldecott, K.W. DNA strand break repair and human genetic disease. Annu. Rev. Genomics Hum. Genet. 8, 37–55 (2007).

    Article  CAS  Google Scholar 

  2. Wyman, C. & Kanaar, R. DNA double-strand break repair: all's well that ends well. Annu. Rev. Genet. 40, 363–383 (2006).

    Article  CAS  Google Scholar 

  3. Audebert, M., Salles, B. & Calsou, P. Involvement of poly(ADP-ribose) polymerase-1 and XRCC1/DNA ligase III in an alternative route for DNA double-strand breaks rejoining. J. Biol. Chem. 279, 55117–55126 (2004).

    Article  CAS  Google Scholar 

  4. Corneo, B. et al. Rag mutations reveal robust alternative end joining. Nature 449, 483–486 (2007).

    Article  CAS  Google Scholar 

  5. Soulas-Sprauel, P. et al. Role for DNA repair factor XRCC4 in immunoglobulin class switch recombination. J. Exp. Med. 204, 1717–1727 (2007).

    Article  CAS  Google Scholar 

  6. Wang, H. et al. DNA ligase III as a candidate component of backup pathways of nonhomologous end joining. Cancer Res. 65, 4020–4030 (2005).

    Article  CAS  Google Scholar 

  7. Yan, C.T. et al. IgH class switching and translocations use a robust non-classical end-joining pathway. Nature 449, 478–482 (2007).

    Article  CAS  Google Scholar 

  8. Han, L. & Yu, K. Altered kinetics of nonhomologous end joining and class switch recombination in ligase IV–deficient B cells. J. Exp. Med. 205, 2745–2753 (2008).

    Article  CAS  Google Scholar 

  9. Buis, J. et al. Mre11 nuclease activity has essential roles in DNA repair and genomic stability distinct from ATM activation. Cell 135, 85–96 (2008).

    Article  CAS  Google Scholar 

  10. Luo, G. et al. Disruption of mRad50 causes embryonic stem cell lethality, abnormal embryonic development, and sensitivity to ionizing radiation. Proc. Natl. Acad. Sci. USA 96, 7376–7381 (1999).

    Article  CAS  Google Scholar 

  11. Zhu, J., Petersen, S., Tessarollo, L. & Nussenzweig, A. Targeted disruption of the Nijmegen breakage syndrome gene NBS1 leads to early embryonic lethality in mice. Curr. Biol. 11, 105–109 (2001).

    Article  CAS  Google Scholar 

  12. Berkovich, E., Monnat, R.J. Jr. & Kastan, M.B. Roles of ATM and NBS1 in chromatin structure modulation and DNA double-strand break repair. Nat. Cell Biol. 9, 683–690 (2007).

    Article  CAS  Google Scholar 

  13. Lisby, M., Barlow, J.H., Burgess, R.C. & Rothstein, R. Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins. Cell 118, 699–713 (2004).

    Article  CAS  Google Scholar 

  14. Shroff, R. et al. Distribution and dynamics of chromatin modification induced by a defined DNA double-strand break. Curr. Biol. 14, 1703–1711 (2004).

    Article  CAS  Google Scholar 

  15. Williams, R.S., Williams, J.S. & Tainer, J.A. Mre11-Rad50-Nbs1 is a keystone complex connecting DNA repair machinery, double-strand break signaling, and the chromatin template. Biochem. Cell Biol. 85, 509–520 (2007).

    Article  CAS  Google Scholar 

  16. Lavin, M.F. ATM and the Mre11 complex combine to recognize and signal DNA double-strand breaks. Oncogene 26, 7749–7758 (2007).

    Article  CAS  Google Scholar 

  17. Lee, J.H. & Paull, T.T. Activation and regulation of ATM kinase activity in response to DNA double-strand breaks. Oncogene 26, 7741–7748 (2007).

    Article  CAS  Google Scholar 

  18. Shiloh, Y. ATM and related protein kinases: safeguarding genome integrity. Nat. Rev. Cancer 3, 155–168 (2003).

    Article  CAS  Google Scholar 

  19. Honjo, T., Muramatsu, M. & Fagarasan, S. AID: how does it aid antibody diversity? Immunity 20, 659–668 (2004).

    Article  CAS  Google Scholar 

  20. Stavnezer, J., Guikema, J.E. & Schrader, C.E. Mechanism and regulation of class switch recombination. Annu. Rev. Immunol. 26, 261–292 (2008).

    Article  CAS  Google Scholar 

  21. Rickert, R.C., Roes, J. & Rajewsky, K. B lymphocyte-specific, Cre-mediated mutagenesis in mice. Nucleic Acids Res. 25, 1317–1318 (1997).

    Article  CAS  Google Scholar 

  22. Lumsden, J.M. et al. Immunoglobulin class switch recombination is impaired in Atm-deficient mice. J. Exp. Med. 200, 1111–1121 (2004).

    Article  CAS  Google Scholar 

  23. Reina-San-Martin, B., Chen, H.T., Nussenzweig, A. & Nussenzweig, M.C. ATM is required for efficient recombination between immunoglobulin switch regions. J. Exp. Med. 200, 1103–1110 (2004).

    Article  CAS  Google Scholar 

  24. Kracker, S. et al. Nibrin functions in Ig class-switch recombination. Proc. Natl. Acad. Sci. USA 102, 1584–1589 (2005).

    Article  CAS  Google Scholar 

  25. Reina-San-Martin, B., Nussenzweig, M.C., Nussenzweig, A. & Difilippantonio, S. Genomic instability, endoreduplication, and diminished Ig class-switch recombination in B cells lacking Nbs1. Proc. Natl. Acad. Sci. USA 102, 1590–1595 (2005).

    Article  CAS  Google Scholar 

  26. Kraus, M., Alimzhanov, M.B., Rajewsky, N. & Rajewsky, K. Survival of resting mature B lymphocytes depends on BCR signaling via the Igα/β heterodimer. Cell 117, 787–800 (2004).

    Article  CAS  Google Scholar 

  27. Franco, S. et al. H2AX prevents DNA breaks from progressing to chromosome breaks and translocations. Mol. Cell 21, 201–214 (2006).

    Article  CAS  Google Scholar 

  28. Rogakou, E.P., Pilch, D.R., Orr, A.H., Ivanova, V.S. & Bonner, W.M. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. Chem. 273, 5858–5868 (1998).

    Article  CAS  Google Scholar 

  29. Stucki, M. et al. MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks. Cell 123, 1213–1226 (2005).

    Article  CAS  Google Scholar 

  30. Lou, Z. et al. MDC1 maintains genomic stability by participating in the amplification of ATM-dependent DNA damage signals. Mol. Cell 21, 187–200 (2006).

    Article  CAS  Google Scholar 

  31. Manis, J.P. et al. 53BP1 links DNA damage-response pathways to immunoglobulin heavy chain class-switch recombination. Nat. Immunol. 5, 481–487 (2004).

    Article  CAS  Google Scholar 

  32. Reina-San-Martin, B. et al. H2AX is required for recombination between immunoglobulin switch regions but not for intra-switch region recombination or somatic hypermutation. J. Exp. Med. 197, 1767–1778 (2003).

    Article  CAS  Google Scholar 

  33. Ward, I.M. et al. 53BP1 is required for class switch recombination. J. Cell Biol. 165, 459–464 (2004).

    Article  CAS  Google Scholar 

  34. Williams, R.S. et al. Mre11 dimers coordinate DNA end bridging and nuclease processing in double-strand-break repair. Cell 135, 97–109 (2008).

    Article  CAS  Google Scholar 

  35. Cahill, D. & Carney, J.P. Dimerization of the Rad50 protein is independent of the conserved hook domain. Mutagenesis 22, 269–274 (2007).

    Article  CAS  Google Scholar 

  36. Hopfner, K.P. et al. The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair. Nature 418, 562–566 (2002).

    Article  CAS  Google Scholar 

  37. Wiltzius, J.J., Hohl, M., Fleming, J.C. & Petrini, J.H. The Rad50 hook domain is a critical determinant of Mre11 complex functions. Nat. Struct. Mol. Biol. 12, 403–407 (2005).

    Article  CAS  Google Scholar 

  38. Moreno-Herrero, F. et al. Mesoscale conformational changes in the DNA-repair complex Rad50/Mre11/Nbs1 upon binding DNA. Nature 437, 440–443 (2005).

    Article  CAS  Google Scholar 

  39. Bressan, D.A., Baxter, B.K. & Petrini, J.H. The Mre11-Rad50-Xrs2 protein complex facilitates homologous recombination-based double-strand break repair in Saccharomyces cerevisiae. Mol. Cell. Biol. 19, 7681–7687 (1999).

    Article  CAS  Google Scholar 

  40. Tauchi, H. et al. Nbs1 is essential for DNA repair by homologous recombination in higher vertebrate cells. Nature 420, 93–98 (2002).

    Article  CAS  Google Scholar 

  41. Lengsfeld, B.M., Rattray, A.J., Bhaskara, V., Ghirlando, R. & Paull, T.T. Sae2 is an endonuclease that processes hairpin DNA cooperatively with the Mre11/Rad50/Xrs2 complex. Mol. Cell 28, 638–651 (2007).

    Article  CAS  Google Scholar 

  42. Sartori, A.A. et al. Human CtIP promotes DNA end resection. Nature 450, 509–514 (2007).

    Article  CAS  Google Scholar 

  43. Paull, T.T. & Gellert, M. The 3′ to 5′ exonuclease activity of Mre 11 facilitates repair of DNA double-strand breaks. Mol. Cell 1, 969–979 (1998).

    Article  CAS  Google Scholar 

  44. Paull, T.T. & Gellert, M. A mechanistic basis for Mre11-directed DNA joining at microhomologies. Proc. Natl. Acad. Sci. USA 97, 6409–6414 (2000).

    Article  CAS  Google Scholar 

  45. Deriano, L., Stracker, T.H., Baker, A., Petrini, J.H. & Roth, D.B. Roles for NBS1 in alternative nonhomologous end-joining of V(D)J recombination intermediates. Mol. Cell 34, 13–25 (2009).

    Article  CAS  Google Scholar 

  46. Helmink, B.A. et al. MRN complex function in the repair of chromosomal Rag-mediated DNA double-strand breaks. J. Exp. Med. 206, 669–679 (2009).

    Article  CAS  Google Scholar 

  47. Deng, Y., Guo, X., Ferguson, D.O. & Chang, S. Multiple roles for Mre11 at uncapped telomeres. Nature advance online publication, doi:10.1038/nature08196. (26 July 2009).

    Article  CAS  Google Scholar 

  48. Xie, A., Kwok, A. & Scully, R. Role of mammalian Mre11 in classical and alternative nonhomologous end joining. Nat. Struct. Mol. Biol. advance online publication, doi:10.1038/nsmb.1640 (26 July 2009).

  49. Rass, E. et al. Role of MRE11 in chromosomal nonhomologous end joining in mammalian cells. Nat. Struct. Mol. Biol. advance online publication, doi: 10.1038/nsmb.1641 (26 July 2009).

  50. Difilippantonio, S. et al. 53BP1 facilitates long-range DNA end joining during V(D)J recombination. Nature 456, 529–533 (2008).

    Article  CAS  Google Scholar 

  51. Dimitrova, N., Chen, Y.C., Spector, D.L. & de Lange, T. 53BP1 promotes non-homologous end joining of telomeres by increasing chromatin mobility. Nature 456, 524–528 (2008).

    Article  CAS  Google Scholar 

  52. Chaudhuri, J. et al. Transcription-targeted DNA deamination by the AID antibody diversification enzyme. Nature 422, 726–730 (2003).

    Article  CAS  Google Scholar 

  53. Begum, N.A. et al. Requirement of non-canonical activity of uracil DNA glycosylase for class switch recombination. J. Biol. Chem. 282, 731–742 (2007).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Support for this work was provided by the US National Institutes of Health (R01-HL079118), the Sidney Kimmel Cancer Research Foundation and the University of Michigan Cancer Center Support Grant 5-P30-CA46592. J.B. is supported by the Cancer Biology Training Program (T32 CA 009676-16) of the University of Michigan Cancer Center. Special thanks to J. Chaudhuri (Sloan-Kettering Institute) for anti-AID antibody, and to W. Dunnick, G. Dressler and D. Lombard for advice on the manuscript and experiments.

Author information

Author notes

  1. Maria Dinkelmann and Elizabeth Spehalski: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pathology, The University of Michigan Medical School, Ann Arbor, Michigan, USA

    Maria Dinkelmann, Elizabeth Spehalski, Trina Stoneham, Jeffrey Buis, Yipin Wu & David O Ferguson

  2. Departments of Internal Medicine and Human Genetics, The University of Michigan, Ann Arbor, Michigan, USA

    JoAnn M Sekiguchi

Authors
  1. Maria Dinkelmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Elizabeth Spehalski
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Trina Stoneham
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jeffrey Buis
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yipin Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. JoAnn M Sekiguchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. David O Ferguson
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.D. generated mouse reagents through complex mouse breedings, planned, performed and analyzed class switch recombination experiments via flow cytometry and performed PCR genotyping; E.S. planned, performed and analyzed class switch recombination experiments, two-color FISH experiments and western blots; T.S. cloned and analyzed DNA sequences of CSR joins and performed and analyzed ELISAs; J.B. planned and analyzed MDC1 foci experiments; Y.W. planned and analyzed H2AX and 53bp1 experiments; J.M.S. planned experiments and analyzed flow cytometric data from bone marrow progenitors; D.O.F. planned the studies and analyzed and interpreted the data.

Corresponding author

Correspondence to David O Ferguson.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 (PDF 3087 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dinkelmann, M., Spehalski, E., Stoneham, T. et al. Multiple functions of MRN in end-joining pathways during isotype class switching. Nat Struct Mol Biol 16, 808–813 (2009). https://doi.org/10.1038/nsmb.1639

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1639

This article is cited by

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