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Ablation of XRCC2/3 transforms immunoglobulin V gene conversion into somatic hypermutation

Nature volume 412, pages 921–926 (2001)Cite this article

Abstract

After gene rearrangement, immunoglobulin V genes are further diversified by either somatic hypermutation or gene conversion1. Hypermutation (in man and mouse) occurs by the fixation of individual, non-templated nucleotide substitutions. Gene conversion (in chicken) is templated by a set of upstream V pseudogenes. Here we show that if the RAD51 paralogues2 XRCC2, XRCC3 or RAD51B are ablated the pattern of diversification of the immunoglobulin V gene in the chicken DT40 B-cell lymphoma line3 exhibits a marked shift from one of gene conversion to one of somatic hypermutation. Non-templated, single-nucleotide substitutions are incorporated at high frequency specifically into the V domain, largely at G/C and with a marked hotspot preference. These mutant DT40 cell lines provide a tractable model for the genetic dissection of immunoglobulin hypermutation and the results support the idea that gene conversion and somatic hypermutation constitute distinct pathways for processing a common lesion in the immunoglobulin V gene. The marked induction of somatic hypermutation that is achieved by ablating the RAD51 paralogues is probably a consequence of modifying the recombination-mediated repair of such initiating lesions.

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Figure 1: Generation of sIgM-loss variants by wild-type and repair-deficient DT40 cells.
Figure 2: Analysis of Vλ sequences cloned from sIgM-loss variants.
Figure 3: Analysis of immunoglobulin sequences cloned from unsorted DT40 populations after 1 month of clonal expansion.
Figure 4: Analysis of sIgM-loss variants in DT40 cells deficient in DNA-PK, Ku70 and RAD51B.

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References

  1. Honjo, T. & Alt, F. W. Immunoglobulin Genes (Academic, London, 1995).

    Google Scholar 

  2. Takata, M. et al. Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs. Mol. Cell. Biol. 21, 2858–2866 (2001).

    Article  CAS  Google Scholar 

  3. Baba, T. W., Giroir, B. P. & Humphries, E. H. Cell lines derived from avian lymphomas exhibit two distinct phenotypes. Virology 144, 139–151 (1985).

    Article  CAS  Google Scholar 

  4. Buerstedde, J. M. et al. Light chain gene conversion continues at high rate in an ALV-induced cell line. EMBO J. 9, 921–927 (1990).

    Article  CAS  Google Scholar 

  5. Kim, S., Humphries, E. H., Tjoelker, L., Carlson, L. & Thompson, C. B. Ongoing diversification of the rearranged immunoglobulin light-chain gene in a bursal lymphoma cell line. Mol. Cell. Biol. 10, 3224–3231 (1990).

    Article  CAS  Google Scholar 

  6. Sayegh, C. E., Drury, G. & Ratcliffe, M. J. Efficient antibody diversification by gene conversion in vivo in the absence of selection for V(D)J-encoded determinants. EMBO J. 18, 6319–6328 (1999).

    Article  CAS  Google Scholar 

  7. Bezzubova, O., Silbergleit, A., Yamaguchi-Iwai, Y., Takeda, S. & Buerstedde, J. M. Reduced X-ray resistance and homologous recombination frequencies in a RAD54-/- mutant of the chicken DT40 cell line. Cell 89, 185–193 (1997).

    Article  CAS  Google Scholar 

  8. Yamaguchi-Iwai, Y. et al. Homologous recombination, but not DNA repair, is reduced in vertebrate cells deficient in RAD52. Mol. Cell. Biol. 18, 6430–6435 (1998).

    Article  CAS  Google Scholar 

  9. Liu, N. et al. XRCC2 and XRCC3, new human Rad51-family members, promote chromosome stability and protect against DNA cross-links and other damages. Mol. Cell 1, 783–793 (1998).

    Article  CAS  Google Scholar 

  10. Johnson, R. D., Liu, N. & Jasin, M. Mammalian XRCC2 promotes the repair of DNA double-strand breaks by homologous recombination. Nature 401, 397–399 (1999).

    CAS  PubMed  Google Scholar 

  11. Pierce, A. J., Johnson, R. D., Thompson, L. H. & Jasin, M. XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. Genes Dev. 13, 2633–2638 (1999).

    Article  CAS  Google Scholar 

  12. Brenneman, M. A., Weiss, A. E., Nickoloff, J. A. & Chen, D. J. XRCC3 is required for efficient repair of chromosome breaks by homologous recombination. Mutat. Res. 459, 89–97 (2000).

    Article  CAS  Google Scholar 

  13. Cui, X. et al. The XRCC2 and XRCC3 repair genes are required for chromosome stability in mammalian cells. Mutat. Res. 434, 75–88 (1999).

    Article  CAS  Google Scholar 

  14. Deans, B., Griffin, C. S., Maconochie, M. & Thacker, J. Xrcc2 is required for genetic stability, embryonic neurogenesis and viability in mice. EMBO J. 19, 6675–6685 (2000).

    Article  CAS  Google Scholar 

  15. Griffin, C. S., Simpson, P. J., Wilson, C. R. & Thacker, J. Mammalian recombination-repair genes XRCC2 and XRCC3 promote correct chromosome segregation. Nature Cell Biol. 2, 757–761 (2000).

    Article  CAS  Google Scholar 

  16. 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 

  17. Rada, C., Ehrenstein, M. R., Neuberger, M. S. & Milstein, C. Hot spot focusing of somatic hypermutation in MSH2-deficient mice suggests two stages of mutational targeting. Immunity 9, 135–141 (1998).

    Article  CAS  Google Scholar 

  18. Diaz, M., Flajnik, M. F. & Klinman, N. Evolution and the molecular basis of somatic hypermutation of antigen receptor genes. Phil. Trans. R. Soc. Lond. B 356, 67–72 (2001).

    Article  CAS  Google Scholar 

  19. 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 

  20. Maizels, N. Somatic hypermutation: how many mechanisms diversify V region sequences? Cell 83, 9–12 (1995).

    Article  CAS  Google Scholar 

  21. Weill, J. C. & Reynaud, C. A. Rearrangement/hypermutation/gene conversion: when, where and why? Immunol. Today 17, 92–97 (1996).

    Article  CAS  Google Scholar 

  22. Xu, B. & Selsing, E. Analysis of sequence transfers resembling gene conversion in a mouse antibody transgene. Science 265, 1590–1593 (1994).

    Article  CAS  Google Scholar 

  23. 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  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  25. Takata, M. et al. The Rad51 paralog Rad51B promotes homologous recombinational repair. Mol. Cell. Biol. 20, 6476–6482 (2000).

    Article  CAS  Google Scholar 

  26. Takata, M. et al. Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. EMBO J. 17, 5497–5508 (1998).

    Article  CAS  Google Scholar 

  27. Bonfield, J. K., Smith, K. & Staden, R. A new DNA sequence assembly program. Nucleic Acids Res. 23, 4992–4999 (1995).

    Article  CAS  Google Scholar 

  28. Reynaud, C. A., Anquez, V., Grimal, H. & Weill, J. C. A hyperconversion mechanism generates the chicken light chain preimmune repertoire. Cell 48, 379–388 (1987).

    Article  CAS  Google Scholar 

  29. McCormack, W. T., Hurley, E. A. & Thompson, C. B. Germ line maintenance of the pseudogene donor pool for somatic immunoglobulin gene conversion in chickens. Mol. Cell. Biol. 13, 821–830 (1993).

    Article  CAS  Google Scholar 

  30. Reynaud, C. A., Dahan, A., Anquez, V. & Weill, J. C. Somatic hyperconversion diversifies the single VH gene of the chicken with a high incidence in the D region. Cell 59, 171–183 (1989).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank A. Johnson for cell sorting and C. Milstein for helpful discussions.

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Authors and Affiliations

  1. Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK

    Julian E. Sale, Daniella M. Calandrini & Michael S. Neuberger

  2. Immunology and Molecular Genetics, Kawasaki Medical School, 577 Matsushima, Kurashiki, 701-0192, Okayama, Japan

    Minoru Takata

  3. CREST Research Project, Radiation Genetics, Faculty of Medicine, Kyoto University, Sakyo-ku, 606-8501, Kyoto, Japan

    Shunichi Takeda

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  1. Julian E. Sale
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  5. Michael S. Neuberger
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Correspondence to Julian E. Sale.

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Sale, J., Calandrini, D., Takata, M. et al. Ablation of XRCC2/3 transforms immunoglobulin V gene conversion into somatic hypermutation. Nature 412, 921–926 (2001). https://doi.org/10.1038/35091100

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