Transcription-targeted DNA deamination by the AID antibody diversification enzyme

Abstract

Activation-induced cytidine deaminase (AID), which is specific to B lymphocytes, is required for class switch recombination (CSR)—a process mediating isotype switching of immunoglobulin—and somatic hypermutation—the introduction of many point mutations into the immunoglobulin variable region genes1,2. It has been suggested that AID may function as an RNA-editing enzyme3 or as a cytidine deaminase on DNA4,5. However, the precise enzymatic activity of AID has not been assessed in previous studies. Similarly, although transcription of the target immunoglobulin locus sequences is required for both CSR and somatic hypermutation, the precise role of transcription has remained speculative6,7,8,9. Here we use two different assays to demonstrate that AID can deaminate specifically cytidines on single-stranded (ss)DNA but not double-stranded (ds)DNA substrates in vitro. However, dsDNA can be deaminated by AID in vitro when the reaction is coupled to transcription. Moreover, a synthetic dsDNA sequence, which targets CSR in vivo in a manner dependent on transcriptional orientation10, was deaminated by AID in vitro with the same transcriptional-orientation-dependence as observed for endogenous CSR. We conclude that transcription targets the DNA deamination activity of AID to dsDNA by generating secondary structures that provide ssDNA substrates.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: DNA cytidine deaminase activity in AID-expressing cells.
Figure 2: ssDNA cytidine deaminase activity co-sediments with AID in glycerol gradients.
Figure 3: Immunoprecipitated AID contains ssDNA cytidine deaminase activity.
Figure 4: Transcription-coupled dsDNA deamination by AID.

References

  1. 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)

    CAS  Article  Google Scholar 

  2. 2

    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)

    CAS  Article  Google Scholar 

  3. 3

    Doi, T., Kinoshita, K., Ikegawa, M., Muramatsu, M. & Honjo, T. De novo protein synthesis is required for the activation-induced cytidine deaminase function in class-switch recombination. Proc. Natl Acad. Sci. USA 18, 2634–2638 (2003)

    ADS  Article  Google Scholar 

  4. 4

    Petersen-Mahrt, S. K., Harris, R. S. & Neuberger, M. S. AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification. Nature 418, 99–103 (2002)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Rada, C. et al. Immunoglobulin isotype switching is inhibited and somatic hypermutation perturbed in UNG-deficient mice. Curr. Biol. 12, 1748–1755 (2002)

    CAS  Article  Google Scholar 

  6. 6

    Manis, J. P., Tian, M. & Alt, F. W. Mechanism and control of class-switch recombination. Trends Immunol. 23, 31–39 (2002)

    CAS  Article  Google Scholar 

  7. 7

    Honjo, T., Kinoshita, K. & Muramatsu, M. Molecular mechanism of class switch recombination: linkage with somatic hypermutation. Annu. Rev. Immunol. 20, 165–196 (2002)

    CAS  Article  Google Scholar 

  8. 8

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

    CAS  Article  Google Scholar 

  9. 9

    Papavasiliou, F. N. & Schatz, D. G. Somatic hypermutation of immunoglobulin genes: merging mechanisms for genetic diversity. Cell 109 (suppl.), 35–44 (2002)

    Article  Google Scholar 

  10. 10

    Shinkura, R. et al. The effect of transcriptional orientation on endogenous switch region function. Nature Immunol. (in the press)

  11. 11

    Bassing, C. H., Swat, W. & Alt, F. W. The mechanism and regulation of chromosomal V(D)J recombination. Cell 109 (suppl.), 45–55 (2002)

    Article  Google Scholar 

  12. 12

    Luby, T. M., Schrader, C. E., Stavnezer, J. & Selsing, E. The mu switch region tandem repeats are important, but not required, for antibody class switch recombination. J. Exp. Med. 193, 159–168 (2001)

    CAS  Article  Google Scholar 

  13. 13

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

    CAS  PubMed  Google Scholar 

  14. 14

    Martin, A. et al. Activation-induced cytidine deaminase turns on somatic hypermutation in hybridomas. Nature 415, 802–806 (2002)

    CAS  Article  Google Scholar 

  15. 15

    Yoshikawa, K. et al. AID enzyme-induced hypermutation in an actively transcribed gene in fibroblasts. Science 296, 2033–2036 (2002)

    ADS  CAS  Article  Google Scholar 

  16. 16

    Di Noia, J. & Neuberger, M. S. Altering the pathway of immunoglobulin hypermutation by inhibiting uracil-DNA glycosylase. Nature 419, 43–48 (2002)

    ADS  CAS  Article  Google Scholar 

  17. 17

    Papavasiliou, F. N. & Schatz, D. G. The activation-induced deaminase functions in a postcleavage step of the somatic hypermutation process. J. Exp. Med. 195, 1193–1198 (2002)

    CAS  Article  Google Scholar 

  18. 18

    Muramatsu, M. et al. Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells. J. Biol. Chem. 274, 18470–18476 (1999)

    CAS  Article  Google Scholar 

  19. 19

    Reaban, M. E. & Griffin, J. A. Induction of RNA-stabilized DNA conformers by transcription of an immunoglobulin switch region. Nature 348, 342–344 (1990)

    ADS  CAS  Article  Google Scholar 

  20. 20

    Daniels, G. A. & Lieber, M. R. RNA:DNA complex formation upon transcription of immunoglobulin switch regions: implications for the mechanism and regulation of class switch recombination. Nucleic Acids Res. 23, 5006–5011 (1995)

    CAS  Article  Google Scholar 

  21. 21

    Tian, M. & Alt, F. W. Transcription-induced cleavage of immunoglobulin switch regions by nucleotide excision repair nucleases in vitro. J. Biol. Chem. 275, 24163–24172 (2000)

    CAS  Article  Google Scholar 

  22. 22

    Mizuta, R. et al. Molecular visualization of immunoglobulin switch region RNA/DNA complex by atomic force microscope. J. Biol. Chem. 278, 4431–4434 (2003)

    CAS  Article  Google Scholar 

  23. 23

    Tashiro, J., Kinoshita, K. & Honjo, T. Palindromic but not G-rich sequences are targets of class switch recombination. Int. Immunol. 13, 495–505 (2001)

    CAS  Article  Google Scholar 

  24. 24

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

    CAS  Article  Google Scholar 

  25. 25

    Shanmugam, A., Shi, M. J., Yauch, L., Stavnezer, J. & Kenter, A. L. Evidence for class-specific factors in immunoglobulin isotype switching. J. Exp. Med. 191, 1365–1380 (2000)

    CAS  Article  Google Scholar 

  26. 26

    Faili, A. et al. AID-dependent somatic hypermutation occurs as a DNA single-strand event in the BL2 cell line. Nature Immunol. 3, 815–821 (2002)

    CAS  Article  Google Scholar 

  27. 27

    Artsimovitch, I. & Landick, R. The transcriptional regulator RfaH stimulates RNA chain synthesis after recruitment to elongation complexes by the exposed nontemplate DNA strand. Cell 109, 193–203 (2002)

    CAS  Article  Google Scholar 

  28. 28

    Storb, U. et al. Cis-acting sequences that affect somatic hypermutation of Ig genes. Immunol. Rev. 162, 153–160 (1998)

    CAS  Article  Google Scholar 

  29. 29

    Yu, K., Chedin, F., Hsieh, C. L., Wilson, T. E. & Lieber, M. R. R-loops at immunoglobulin class switch regions within the chromosomes of stimulated B cells. Nature Immunol. (in the press)

  30. 30

    Sartori, A. A., Schar, P., Fitz-Gibbon, S., Miller, J. H. & Jiricny, J. Biochemical characterization of uracil processing activities in the hyperthermophilic archaeon Pyrobaculum aerophilum. J. Biol. Chem. 276, 29979–29986 (2001)

    CAS  Article  Google Scholar 

  31. 31

    Ramiro, A. R., Stavropoulos, P., Jankovic, M. & Nussenzweig, M. C. Transcription enhances AID-mediated cytidine deamination by exposing ssDNA on the nontemplate strand. Nature Immunol. (in the press)

Download references

Acknowledgements

We thank T. Honjo for providing AID-deficient mice and B. Demple for helpful discussions. We also thank J. Manis, A. Zarrin, S. Ranganath and S. Saleque for suggestions and critical reading of the manuscript. This work was supported by a NIH grant (F.W.A.) and a NIH training grant (M.T.). K.C. is supported by a Pfizer Postdoctoral Fellowship in Rheumatology/Immunology and E.P. is supported by a Fondation pour la Recherche Medicale Postdoctoral Fellowship. F.W.A. is an Investigator and J.C. an Associate of the Howard Hughes Medical Institute.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Frederick W. Alt.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Chaudhuri, J., Tian, M., Khuong, C. et al. Transcription-targeted DNA deamination by the AID antibody diversification enzyme. Nature 422, 726–730 (2003). https://doi.org/10.1038/nature01574

Download citation

Further reading

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.