Article | Published:

Altered somatic hypermutation and reduced class-switch recombination in exonuclease 1–mutant mice

Abstract

The generation of protective antibodies requires somatic hypermutation (SHM) and class-switch recombination (CSR) of immunoglobulin genes. Here we show that mice mutant for exonuclease 1 (Exo1), which participates in DNA mismatch repair (MMR), have decreased CSR and changes in the characteristics of SHM similar to those previously observed in mice mutant for the MMR protein Msh2. Exo1 is thus the first exonuclease shown to be involved in SHM and CSR. The phenotype of Exo1−/− mice and the finding that Exo1 and Mlh1 are physically associated with mutating variable regions support the idea that Exo1 and MMR participate directly in SHM and CSR.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1

    MacLennan, I.C. Germinal centers. Annu. Rev. Immunol. 12, 117–139 (1994).

  2. 2

    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).

  3. 3

    Stavnezer, J. Molecular processes that regulate class switching. Curr. Top. Microbiol. Immunol. 245, 127–168 (2000).

  4. 4

    Storb, U. Progress in understanding the mechanism and consequences of somatic hypermutation. Immunol. Rev. 162, 5–11 (1998).

  5. 5

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

  6. 6

    Martin, A. & Scharff, M.D. AID and mismatch repair in antibody diversification. Nat. Rev. Immunol. 2, 605–614 (2002).

  7. 7

    Bransteitter, R., Pham, P., Scharff, M.D. & Goodman, M.F. Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of RNase. Proc. Natl. Acad. Sci. USA 100, 4102–4107 (2003).

  8. 8

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

  9. 9

    Dickerson, S.K., Market, E., Besmer, E. & Papavasiliou, F.N. AID mediates hypermutation by deaminating single stranded DNA. J. Exp. Med. 197, 1291–1296 (2003).

  10. 10

    Sohail, A., Klapacz, J., Samaranayake, M., Ullah, A. & Bhagwat, A.S. Human activation-induced cytidine deaminase causes transcription-dependent, strand-biased C to U deaminations. Nucleic Acids Res. 31, 2990–2994 (2003).

  11. 11

    Shinkura, R. et al. The influence of transcriptional orientation on endogenous switch region function. Nat. Immunol. 4, 435–441 (2003).

  12. 12

    Yu, K., Chedin, F., Hsieh, C.L., Wilson, T.E. & Lieber, M.R. R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells. Nat. Immunol. 4, 442–451 (2003).

  13. 13

    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).

  14. 14

    Zeng, X. et al. DNA polymerase eta is an A-T mutator in somatic hypermutation of immunoglobulin variable genes. Nat. Immunol. 2, 537–541 (2001).

  15. 15

    Wiesendanger, M., Kneitz, B., Edelmann, W. & Scharff, M.D. Somatic mutation in MSH3, MSH6, and MSH3/MSH6-deficient mice reveals a role for the MSH2-MSH6 heterodimer in modulating the base substitution pattern. J. Exp. Med. 191, 579–584 (2000).

  16. 16

    Buermeyer, A.B., Deschenes, S.M., Baker, S.M. & Liskay, R.M. Mammalian DNA mismatch repair. Annu. Rev. Genet. 33, 533–564 (1999).

  17. 17

    Evans, E. & Alani, E. Roles for mismatch repair factors in regulating genetic recombination. Mol. Cell Biol. 20, 7839–7844 (2000).

  18. 18

    Marsischky, G.T., Filosi, N., Kane, M.F. & Kolodner, R. Redundancy of Saccharomyces cerevisiae MSH3 and MSH6 in MSH2-dependent mismatch repair. Genes Dev. 10, 407–420 (1996).

  19. 19

    Palombo, F. et al. GTBP, a 160-kilodalton protein essential for mismatch-binding activity in human cells. Science 268, 1912–1914 (1995).

  20. 20

    Kolodner, R. Biochemistry and genetics of eukaryotic mismatch repair. Genes Dev. 10, 1433–1442 (1996).

  21. 21

    Szankasi, P. & Smith, G.R. A role for exonuclease I from S. pombe in mutation avoidance and mismatch correction. Science 267, 1166–1169 (1995).

  22. 22

    Genschel, J., Bazemore, L.R. & Modrich, P. Human exonuclease I is required for 5′ and 3′ mismatch repair. J. Biol. Chem. 277, 13302–13311 (2002).

  23. 23

    Tishkoff, D.X. et al. Identification and characterization of Saccharomyces cerevisiae EXO1, a gene encoding an exonuclease that interacts with MSH2. Proc. Natl. Acad. Sci. USA 94, 7487–7492 (1997).

  24. 24

    Tran, P.T., Simon, J.A. & Liskay, R.M. Interactions of Exo1p with components of MutLα in Saccharomyces cerevisiae . Proc. Natl. Acad. Sci. USA 98, 9760–9765 (2001).

  25. 25

    Schmutte, C., Sadoff, M.M., Shim, K.S., Acharya, S. & Fishel, R. The interaction of DNA mismatch repair proteins with human exonuclease I. J. Biol. Chem. 276, 33011–33018 (2001).

  26. 26

    Amin, N.S., Nguyen, M.N., Oh, S. & Kolodner, R.D. exo1-dependent mutator mutations: model system for studying functional interactions in mismatch repair. Mol. Cell Biol. 21, 5142–5155 (2001).

  27. 27

    Wei, K. et al. Inactivation of Exonuclease 1 in mice results in DNA mismatch repair defects, increased cancer susceptibility and male and female sterility. Genes Dev. 17, 603–614 (2003).

  28. 28

    Schrader, C.E., Edelmann, W., Kucherlapati, R. & Stavnezer, J. Reduced isotype switching in splenic B cells from mice deficient in mismatch repair enzymes. J. Exp. Med. 190, 323–330 (1999).

  29. 29

    Ehrenstein, M.R., Rada, C., Jones, A.M., Milstein, C. & Neuberger, M.S. Switch junction sequences in PMS2-deficient mice reveal a microhomology-mediated mechanism of Ig class switch recombination. Proc. Natl. Acad. Sci. USA 98, 14553–14558 (2001).

  30. 30

    Schrader, C.E., Vardo, J. & Stavnezer, J. Role for mismatch repair proteins Msh2, Mlh1, and Pms2 in immunoglobulin class switching shown by sequence analysis of recombination junctions. J. Exp. Med. 195, 367–373 (2002).

  31. 31

    Takahashi, Y., Dutta, P.R., Cerasoli, D.M. & Kelsoe, G. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. V. Affinity maturation develops in two stages of clonal selection. J. Exp. Med. 187, 885–895 (1998).

  32. 32

    Frey, S. et al. Mismatch repair deficiency interferes with the accumulation of mutations in chronically stimulated B cells and not with the hypermutation process. Immunity 9, 127–134 (1998).

  33. 33

    Denepoux, S. et al. Induction of somatic mutation in a human B cell line in vitro . Immunity 6, 35–46 (1997).

  34. 34

    Poltoratsky, V. et al. Expression of error-prone polymerases in BL2 cells activated for Ig somatic hypermutation. Proc. Natl. Acad. Sci. USA 98, 7976–7981 (2001).

  35. 35

    Woo, C.J., Martin, A. & Scharff, M.D. Induction of hypermutation is associated with modifications of variable region chromatin in BL2 cells. Immunity 19, 479–489 (2003).

  36. 36

    Bemark, M. et al. Somatic hypermutation in the absence of DNA-PK or Rag1 activity. J. Exp. Med. 192, 1509–1514 (2000).

  37. 37

    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).

  38. 38

    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).

  39. 39

    Vora, K.A. et al. Severe attenuation of the B cell immune response in Msh2-deficient mice. J. Exp. Med. 189, 471–481 (1999).

  40. 40

    Alabyev, B. & Manser, T. Bcl-2 rescues the germinal center response but does not alter the V gene somatic hypermutation spectrum in MSH2-deficient mice. J. Immunol. 169, 3819–3824 (2002).

  41. 41

    Kim, N., Bozek, G., Lo, J.C. & Storb, U. Different mismatch repair deficiencies all have the same effects on somatic hypermutation: intact primary mechanism accompanied by secondary modifications. J. Exp. Med. 190, 21–30 (1999).

  42. 42

    Winter, D.B. et al. Altered spectra of hypermutation in antibodies from mice deficient for the DNA mismatch repair protein PMS2. Proc. Natl. Acad. Sci. USA 95, 6953–6958 (1998).

  43. 43

    Schrader, C.E., Vardo, J. & Stavnezer, J. Mlh1 can function in antibody class switch recombination independently of Msh2. J. Exp. Med. 197, 1377–1383 (2003).

  44. 44

    Kong, Q. & Maizels, N. DNA breaks in hypermutating immunoglobulin genes: evidence for a break and repair pathway of somatic mutation. Genetics 158, 369–378 (2001).

  45. 45

    Papavasiliou, F.N. & Schatz, D.G. Somatic hypermutation of immunoglobulin genes: merging mechanisms for genetic diversity. Cell 109 (Suppl.), S35–S44 (2002).

  46. 46

    Phung, Q.H. et al. Increased hypermutation at G and C nucleotides in immunoglobulin variable genes from mice deficient in the MSH2 mismatch repair protein. J. Exp. Med. 187, 1745–1751 (1998).

  47. 47

    Ehrenstein, M.R. & Neuberger, M.S. Deficiency in msh2 affects the efficiency and local sequence specificity of immunoglobulin class-switch recombination: parallels with somatic hypermutation. EMBO J. 18, 3484–3490 (1999).

  48. 48

    Wang, T.F., Kleckner, N. & Hunter, N. Functional specificity of MutL homologs in yeast: evidence for three Mlh1-based heterocomplexes with distinct roles during meiosis in recombination and mismatch correction. Proc. Natl. Acad. Sci. USA 96, 13914–13919 (1999).

  49. 49

    Tsubouchi, H. & Ogawa, H. Exo1 roles for repair of DNA double-strand breaks and meiotic crossing over in Saccharomyces cerevisiae . Mol. Biol. Cell 11, 2221–2233 (2000).

  50. 50

    Sack, S.Z., Bardwell, P.D. & Scharff, M.D. Testing the reverse transcriptase model of somatic mutation. Mol. Immunol. 38, 303–311 (2001).

Download references

Acknowledgements

This work was supported by grants from the US National Institutes of Health: 5T32 CA 09173 (P.D.B.), T326 MO 7491 (C.J.W.), CA 76329 and CA 93484 (W.E.), AI 53362, CA102705 and CA72649 (M.D.S.). A.M. and Z.L. are recipients of Cancer Research Institute Fellowships and A.M. is currently a Special Fellow from the Leukemia and Lymphoma Society. W.E. is also supported by the Irma T. Hirschl Career Scientist Award. M.D.S. has the additional support of the Harry Eagle Chair provided by the Women's Division of the Albert Einstein College of Medicine.

Author information

Competing interests

The authors declare no competing financial interests.

Correspondence to Matthew D Scharff.

Supplementary information

Supplementary Fig. 1 (PDF 43 kb)

Rights and permissions

Reprints and Permissions

About this article

Further reading

Figure 1: FACS analysis of in vitro–switched splenocytes.
Figure 2: Serum ELISA of anti-NP immunoglobulin response.
Figure 3: Mutation analysis of the immunoglobulin gene.
Figure 4: Chromatin immunoprecipitation (ChIP) experiments for Exo1 and Mlh1 in the BL2 Burkitt's lymphoma cell line.