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Transposition mediated by RAG1 and RAG2 and its implications for the evolution of the immune system

Nature volume 394, pages 744–751 (1998)Cite this article

Abstract

Immunoglobulin and T-cell-receptor genes are assembled from component gene segments in developing lymphocytes by a site-specific recombination reaction, V (D)J recombination. The proteins encoded by the recombination-activating genes, RAG1 and RAG2, are essential in this reaction, mediating sequence-specific DNA recognition of well-defined recombination signals and DNA cleavage next to these signals. Here we show that RAG1 and RAG2 together form a transposase capable of excising a piece of DNA containing recombination signals from a donor site and inserting it into a target DNA molecule. The products formed contain a short duplication of target DNA immediately flanking the transposed fragment, a structure like that created by retroviral integration and all known transposition reactions. The results support the theory that RAG1 and RAG2 were once components of a transposable element, and that the split nature of immunoglobulin and T-cell-receptor genes derives from germline insertion of this element into an ancestral receptor gene soon after the evolutionary divergence of jawed and jawless vertebrates.

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Figure 1: Time course of cleavage and band X formation.
Figure 2: Stable association of the RAG and HMG2 proteins with band X confers nuclease resistance.
Figure 3: Conditions for band X formation.
Figure 4: Intramolecular transposition events using the 329-bp SE/SE substrate.
Figure 5: Mapping of the 5′ ends of the SE/SE fragment and band X.
Figure 6: Intermolecular transposition events.
Figure 7: RAG-mediated transposition and a model for the origins of split antigen-receptor genes.

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References

  1. Tonegawa, S. Somatic generation of antibody diversity. Nature 302 , 575–581 (1983).

    Article  ADS  CAS  Google Scholar 

  2. Lewis, S. M. The mechanism of V(D)J joining: lessons from molecular, immunological, and comparative analyses. Adv. Immunol. 56, 27–150 (1994).

    Article  CAS  Google Scholar 

  3. Schatz, D. G., Oettinger, M. A. & Baltimore, D. The V(D)J recombination activating gene (RAG-1). Cell 59, 1035–1048 ( 1989).

    Article  CAS  Google Scholar 

  4. Oettinger, M. A., Schatz, D. G., Gorka, C. & Baltimore, D. RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination. Science 248, 1517–1523 ( 1990).

    Article  ADS  CAS  Google Scholar 

  5. McBlane, J. F.et al. Cleavage at a V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps. Cell 83, 387–395 (1995).

    Article  CAS  Google Scholar 

  6. Eastman, Q. M., Leu, T. M. J. & Schatz, D. G. Initiation of V(D)J recombination in vitro obeying the 12/23 rule. Nature 380, 85– 88 (1996).

    Article  ADS  CAS  Google Scholar 

  7. van Gent, D. C., Ramsden, D. A. & Gellert, M. The RAG1 and RAG2 proteins establish the 12/23 rule in V(D)J recombination. Cell 85, 107– 113 (1996).

    Article  CAS  Google Scholar 

  8. Sawchuk, D. J.et al. V(D)J recombination: modulation of RAG1 and RAG2 cleavage activity on 12/23 substrates by whole cell extract and DNA bending proteins. J. Exp. Med. 185, 2025–2032 (1997).

    Article  CAS  Google Scholar 

  9. van Gent, D. C., Hiom, K., Paull, T. T. & Gellert, M. Stimulation of V(D)J cleavage by high mobility group proteins. EMBO J. 16, 2665–2670 (1997).

    Article  CAS  Google Scholar 

  10. van Gent, D. C., Mizuuchi, K. & Gellert, M. Similarities between initiation of V(D)J recombination and retroviral integration. Science 271, 1592–1594 (1996).

    Article  ADS  CAS  Google Scholar 

  11. Weaver, D. T. What to do at an end—DNA double-strand-break repair. Trends Genet. 11, 388–392 ( 1995).

    Article  CAS  Google Scholar 

  12. Chu, G. Double strand break repair. J. Biol. Chem. 272, 24097–24100 (1997).

    Article  CAS  Google Scholar 

  13. Grawunderr, U., West, R. B. & Lieber, M. R. Antigen receptor gene rearrangement. Curr. Opin. Immunol. 10, 172–180 (1998).

    Article  Google Scholar 

  14. Thompson, C. B. New insights into V(D)J recombination and its role in the evolution of the immune system. Immunity 3, 531– 539 (1995).

    Article  CAS  Google Scholar 

  15. Lewis, S. M. & Wu, G. E. The origins of V(D)J recombination. Cell 88, 159–162 (1997)

    Article  CAS  Google Scholar 

  16. Litman, G. W.et al. Phylogenetic diversification of immunoglobulin genes and the antibody repertoire. Mol. Biol. Evol. 10, 60–72 (1993).

    CAS  PubMed  Google Scholar 

  17. Gellert, M. Recent advances in understanding V(D)J recombination. Adv. Immunol. 64, 39–64 ( 1996).

    Article  Google Scholar 

  18. Craig, N. L. Unity in transposition reactions. Science 270, 253–254 (1995).

    Article  ADS  CAS  Google Scholar 

  19. Agrawal, A. & Schatz, D. G. RAG1 and RAG2 form a stable post-cleavage synaptic complex with DNA containing signal ends in V(D)J recombination. Cell 89, 43–53 ( 1997).

    Article  CAS  Google Scholar 

  20. Mizuuchi, K. Transpositional recombination: mechanistic insights from studies of Mu and other elements. Annu. Rev. Biochem. 61, 1011–1051 (1992).

    Article  CAS  Google Scholar 

  21. Melek, M., Gellert, M. & van gent, D. C. Rejoining of DNA by the RAG1 and RAG2 proteins. Science 280, 301–303 ( 1998).

    Article  ADS  CAS  Google Scholar 

  22. Spanopoulou, E.et al . The homeodomain of Rag-1 reveals the parallel mechanisms of bacterial and V(D)J recombination. Cell 87, 263–276 (1996).

    Article  CAS  Google Scholar 

  23. Weinert, T. A., Derbyshire, K. M., Hughson, F. M. & Grindley, N. D. F. Replicative and conservative transpositional recombination of insertion sequences. Cold Spring Harb. Symp. Quant. Biol. 49, 251–260 (1984).

    Article  CAS  Google Scholar 

  24. Benjamin, H. W. & Kleckner, N. Intramolecular transposition by Tn10. Cell 59, 373– 383 (1989).

    Article  CAS  Google Scholar 

  25. Isberg, R. R. & Syvanen, M. Tn5 transposes independently of cointegrate resolution. Evidence for an alternative model for transposition. J. Mol. Biol. 182, 69– 78 (1985).

    Article  CAS  Google Scholar 

  26. Shoemaker, C., Hoffmann, J., Goff, S. P. & Baltimore, D. Intramolecular integration within Moloney Murine Leukemia Virus DNA. J. Virol. 40, 164–172 ( 1981).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Lee, Y. M. H. & Coffin, J. M. Efficient autointegration of avian retrovirus DNA in vitro. J. Virol. 64, 5958 –5965 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Fujiware, T. & Mizuuchi, K. Retroviral DNA integration: structure of an integration intermediate. Cell 54, 497–504 (1988).

    Article  Google Scholar 

  29. Brown, P. O., Bowerman, B., Varmus, H. E. & Bishop, J. M. Retroviral integration: structure of the initial covalent product and its precursor, and a role for the viral IN protein. Proc. Natl Acad. Sci. USA 86, 2525–2529 ( 1989).

    Article  ADS  CAS  Google Scholar 

  30. Berg, D. E. & Howe, M. M. Mobile DNA(Am. Soc. Microbiol., Washington DC, (1989)).

    Google Scholar 

  31. Mizuuchi, K. Polynucleotidyl transfer reactions in transpositional DNA recombination. J. Biol. Chem. 267, 21273–21276 (1992).

    CAS  PubMed  Google Scholar 

  32. Kleckner, N., Chalmers, R. M., Kwon, D., Sakai, J. & Bolland, S. Tn10 and IS10 transposition and chromosome rearrangements: mechanism and regulation in vivo and in vitro. Curr. Top. Microbiol. Immunol. 204, 49–82 (1996).

    CAS  PubMed  Google Scholar 

  33. Hiom, K. & Gellert, M. Assembly of a 12/23 paired signal complex: a critical control point in V(D)J recombination. Mol. Cell 1, 1011–1019 ( 1998).

    Article  CAS  Google Scholar 

  34. Dyda, F.et al. Crystal structure of the catalytic domain of HIV-1 integrase: similarity to other polynucleotidyl transferases. Science 266, 1981–1986 (1994).

    Article  ADS  CAS  Google Scholar 

  35. Rice, P., & Mizuuchi, K. Structure of the bacteriophage Mu transposase core: a common structural motif for DNA transposition and retroviral integration. Cell 82, 209– 220 (1995).

    Article  CAS  Google Scholar 

  36. Bujacz, G.et al. High-resolution structure of the catalytic domain of avian sarcoma virus integrase. J. Mol. Biol. 253, 333– 346 (1995).

    Article  CAS  Google Scholar 

  37. Chalmers, R., Guhathakurta, A., Benjamin, H. & Kleckner, N. IHF modulation of Tn10 transposition: sensory transduction of supercoiling status via a proposed protein/DNA molecular spring. Cell 93, 897–908 (1998).

    Article  CAS  Google Scholar 

  38. Craig, N. L. Target site selection in transposition. Annu.. Rev. Biochem. 66, 437–474 (1997).

    Article  CAS  Google Scholar 

  39. Sakai, J. & Kleckner, N. The Tn10 synaptic complex can capture a target DNA only after transposon excision. Cell 89 , 205–214 (1997).

    Article  CAS  Google Scholar 

  40. Rast, J. P.et al. α, β, γ and δ T cell antigen receptor genes arose early in vertebrate phylogeny. Immunity 6, 1–11 (1997).

    Article  CAS  Google Scholar 

  41. Sakano, H., Hüppi, K., Heinrich, G. & Tonegawa, S. Sequences at the somatic recombination sites of immunoglobulin light-chain genes. Nature 280, 288– 294 (1979).

    Article  ADS  CAS  Google Scholar 

  42. Zwilling, S., König, H. & Wirth, T. High mobility group protein 2 functionally interacts with the POU domains of octamer transcription factors. EMBO J. 14, 1198–1208 ( 1995).

    Article  CAS  Google Scholar 

  43. Lewis, S. M. & Hesse, J. E. Cutting and closing without recombination in V(D)J joining. EMBO J. 10, 3631– 3639 (1991).

    Article  CAS  Google Scholar 

  44. Hsieh, C., McCloskey, R. P., Radany, E. & Lieber, M. R. V(D)J recombination: evidence that a replicative mechanism is not required. Mol. Cell. Biol. 11, 3972– 3977 (1991).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank D. Hesslein for GST-RAG protein; I. Villey for HMG2 and RAG proteins; C.-L. Tsai for RAG proteins; P. Shockett for help with denaturing polyacrylamide gels; W. Stephen for bacterial plates and media; F. Livak for pTetRSS; E. Spanopoulou for the GST–RAG plasmids; T. Wirth for the HMG2 plasmid; J. Repasky and E. Corbett for help with various procedures; N. Craig and G. Litman for comments on the manuscript; and the W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University for oligonucleotides and DNA sequencing. D.G.S. is an associate investigator of the Howard Hughes Medical Institute. This work was supported by a grant from the NIH.

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Authors and Affiliations

  1. Department of Pharmacology, New Haven, 06510, Connecticut, USA

    Alka Agrawal

  2. Department of Molecular Biophysics and Biochemistry, New Haven, 06510 , Connecticut, USA

    Quinn M. Eastman

  3. Howard Hughes Medical Institute, Section of Immunobiology, Yale University School of Medicine, New Haven, 06510, Connecticut, USA

    David G. Schatz

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  1. Alka Agrawal
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Correspondence to David G. Schatz.

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Agrawal, A., Eastman, Q. & Schatz, D. Transposition mediated by RAG1 and RAG2 and its implications for the evolution of the immune system. Nature 394, 744–751 (1998). https://doi.org/10.1038/29457

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