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

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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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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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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