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Replication protein A interacts with AID to promote deamination of somatic hypermutation targets

Nature volume 430, pages 992–998 (2004)Cite this article

Abstract

Activation-induced cytidine deaminase (AID) is a single-stranded (ss) DNA deaminase required for somatic hypermutation (SHM) and class switch recombination of immunoglobulin genes. Class switch recombination involves transcription through switch regions, which generates ssDNA within R loops. However, although transcription through immunoglobulin variable region exons is required for SHM, it does not generate stable ssDNA, which leaves the mechanism of AID targeting unresolved. Here we characterize the mechanism of AID targeting to in-vitro-transcribed substrates harbouring SHM motifs. We show that the targeting activity of AID is due to replication protein A (RPA), a ssDNA-binding protein involved in replication, recombination and repair. The 32-kDa subunit of RPA interacts specifically with AID from activated B cells in a manner that seems to be dependent on post-translational AID modification. Thus, our study implicates RPA as a novel factor involved in immunoglobulin diversification. We propose that B-cell-specific AID–RPA complexes preferentially bind to ssDNA of small transcription bubbles at SHM ‘hotspots’, leading to AID-mediated deamination and RPA-mediated recruitment of DNA repair proteins.

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Figure 1: Transcription-coupled deamination on SHM substrates.
Figure 2: Strand-specific targeting of AID and CF.
Figure 3: AID interacts with CF.
Figure 4: The complementing factor is RPA.
Figure 5: Coordinate binding of AID and RPA to transcribed SHM substrate.
Figure 6: B-cell specificity of the AID–RPA complex.

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References

  1. Jung, D. & Alt, F. W. Unraveling V(D)J recombination: Insights into gene regulation. Cell 116, 299–311 (2004)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Li, Z., Woo, C. J., Iglesias-Ussel, M. D., Ronai, D. & Scharff, M. D. The generation of antibody diversity through somatic hypermutation and class switch recombination. Genes Dev. 18, 1–11 (2004)

    Article  Google Scholar 

  6. Chaudhuri, J. & Alt, F. W. Class switch recombination: interplay of transcription, DNA deamination and DNA repair. Nature Rev. Immunol 4, 541–552 (2004)

    Article  CAS  Google Scholar 

  7. 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 

  8. 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 

  9. Betz, A. G., Neuberger, M. S. & Milstein, C. Discriminating intrinsic and antigen-selected mutational hotspots in immunoglobulin V genes. Immunol. Today 14, 405–411 (1993)

    Article  CAS  Google Scholar 

  10. Neuberger, M. S. et al. Monitoring and interpreting the intrinsic features of somatic hypermutation. Immunol. Rev. 162, 107–116 (1998)

    Article  CAS  Google Scholar 

  11. Storb, U. & Stavnezer, J. Immunoglobulin genes: generating diversity with AID and UNG. Curr. Biol. 12, 725–727 (2002)

    Article  Google Scholar 

  12. Yu, K. & Lieber, M. R. Nucleic acid structures and enzymes in the immunoglobulin class switch recombination mechanism. DNA Repair 2, 1163–1174 (2003)

    Article  CAS  Google Scholar 

  13. Nambu, Y. et al. Transcription-coupled events associating with immunoglobulin switch region chromatin. Science 302, 2137–2140 (2003)

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  15. 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)

    Article  CAS  Google Scholar 

  16. 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)

    Article  ADS  CAS  Google Scholar 

  17. 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)

    Article  CAS  Google Scholar 

  18. 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)

    Article  ADS  CAS  Google Scholar 

  19. 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)

    Article  CAS  Google Scholar 

  20. 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. Nature Immunol. 4, 442–451 (2003)

    Article  CAS  Google Scholar 

  21. Pham, P., Bransteitter, R., Petruska, J. & Goodman, M. F. Processive AID-catalysed cytosine deamination on single-stranded DNA simulates somatic hypermutation. Nature 424, 103–107 (2003)

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  24. Betz, A. G. et al. Elements regulating somatic hypermutation of an immunoglobulin kappa gene: critical role for the intron enhancer/matrix attachment region. Cell 77, 239–248 (1994)

    Article  CAS  Google Scholar 

  25. Peters, A. & Storb, U. Somatic hypermutation of immunoglobulin genes is linked to transcription initiation. Immunity 4, 57–65 (1996)

    Article  CAS  Google Scholar 

  26. Fukita, Y., Jacobs, H. & Rajewsky, K. Somatic hypermutation in the heavy chain locus correlates with transcription. Immunity 9, 105–114 (1998)

    Article  CAS  Google Scholar 

  27. Yelamos, J. et al. Targeting of non-Ig sequences in place of the V segment by somatic hypermutation. Nature 376, 225–229 (1995)

    Article  ADS  CAS  Google Scholar 

  28. 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 

  29. 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 

  30. Michael, N. et al. Effects of sequence and structure on the hypermutability of immunoglobulin genes. Immunity 16, 123–134 (2002)

    Article  CAS  Google Scholar 

  31. Ta, V. T. et al. AID mutant analyses indicate requirement for class-switch-specific cofactors. Nature Immunol. 4, 843–848 (2003)

    Article  CAS  Google Scholar 

  32. Wobbe, C. R. et al. Replication of simian virus 40 origin-containing DNA in vitro with purified proteins. Proc. Natl Acad. Sci. USA 84, 1834–1838 (1987)

    Article  ADS  CAS  Google Scholar 

  33. Wold, M. S. & Kelly, T. Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA. Proc. Natl Acad. Sci. USA 85, 2523–2527 (1988)

    Article  ADS  CAS  Google Scholar 

  34. Fairman, M. P. & Stillman, B. Cellular factors required for multiple stages of SV40 DNA replication in vitro. EMBO J. 7, 1211–1218 (1988)

    Article  CAS  Google Scholar 

  35. Wold, M. S. Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. Annu. Rev. Biochem. 66, 61–92 (1997)

    Article  CAS  Google Scholar 

  36. Yu, K., Huang, F. T. & Lieber, M. R. DNA substrate length and surrounding sequence affect the activation-induced deaminase activity at cytidine. J. Biol. Chem. 279, 6496–6500 (2004)

    Article  CAS  Google Scholar 

  37. Matsunaga, T., Park, C. H., Bessho, T., Mu, D. & Sancar, A. Replication protein A confers structure-specific endonuclease activities to the XPF-ERCC1 and XPG subunits of human DNA repair excision nuclease. J. Biol. Chem. 271, 11047–11050 (1996)

    Article  CAS  Google Scholar 

  38. Nagelhus, T. A. et al. A sequence in the N-terminal region of human uracil-DNA glycosylase with homology to XPA interacts with the C-terminal part of the 34-kDa subunit of replication protein A. J. Biol. Chem. 272, 6561–6566 (1997)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  40. Milstein, C., Neuberger, M. S. & Staden, R. Both DNA strands of antibody genes are hypermutation targets. Proc. Natl Acad. Sci. USA 95, 8791–8794 (1998)

    Article  ADS  CAS  Google Scholar 

  41. Ramiro, A. R., Stavropoulos, P., Jankovic, M. & Nussenzweig, M. C. Transcription enhances AID-mediated cytidine deamination by exposing single-stranded DNA on the nontemplate strand. Nature Immunol. 4, 452–456 (2003)

    Article  CAS  Google Scholar 

  42. 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)

    Article  CAS  Google Scholar 

  43. Steitz, T. A. The structural basis of the transition from initiation to elongation phases of transcription, as well as translocation and strand separation, by T7 RNA polymerase. Curr. Opin. Struct. Biol. 14, 4–9 (2004)

    Article  CAS  Google Scholar 

  44. Bolland, D. J. et al. Antisense intergenic transcription in V(D)J recombination. Nature Immunol. 5, 630–637 (2004)

    Article  CAS  Google Scholar 

  45. Maldonado, E. et al. A human RNA polymerase II complex associated with SRB and DNA-repair proteins. Nature 381, 86–89 (1996)

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  47. Martin, A. & Scharff, M. D. Somatic hypermutation of the AID transgene in B and non-B cells. Proc. Natl Acad. Sci. USA 99, 12304–12308 (2002)

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  50. Henricksen, L. A., Umbricht, C. B. & Wold, M. S. Recombinant replication protein A: expression, complex formation, and functional characterization. J. Biol. Chem. 269, 11121–11132 (1994)

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank T. Honjo for providing AID-deficient mice and B. Stillman for providing bacterial strains expressing recombinant AID. We also thank M. Tian, A. Zarrin, J. Manis and C. Bassing for critical reading of the manuscript, D. Chowdhury for advice on the ChIP experiments, and B. Stillman and S. Elledge for discussions. This work was supported by an NIH grant to F.W.A. F.W.A. is an Investigator of the Howard Hughes Medical Institute.

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  1. Howard Hughes Medical Institute, Children's Hospital, Center for Blood Research and Department of Genetics, Harvard University Medical School, Boston, Massachusetts, 02115, USA

    Jayanta Chaudhuri, Chan Khuong & Frederick W. Alt

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  1. Jayanta Chaudhuri
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  2. Chan Khuong
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Correspondence to Frederick W. Alt.

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Supplementary information

Supplementary Figure 1

Purification of AID. (PPT 2597 kb)

Supplementary Figure 2

The RGYW sequence undergoes preferential deamination. (PPT 62 kb)

Supplementary Figure 3

AID·RPA interaction is not mediated via DNA. (PPT 74 kb)

Supplementary Figure 4

Phosphatase treatment of AID inhibits RPA-mediated dsDNA deaminase activity. (PPT 24 kb)

Supplementary Figure 5

Dephosphorylation of AID impairs interaction with RPA. (PPT 18 kb)

Supplementary Figure 6

Chromatin association of RPA and AID at transcribed S regions. (PPT 1221 kb)

Supplementary Figure 7

Model for RPA/AID activity in SHM and CSR. (PPT 32 kb)

Supplementary Figure legends (DOC 24 kb)

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Chaudhuri, J., Khuong, C. & Alt, F. Replication protein A interacts with AID to promote deamination of somatic hypermutation targets. Nature 430, 992–998 (2004). https://doi.org/10.1038/nature02821

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