Sistema inmune

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Implications of transposition mediated by V(D) J-recombination proteins RAG1 and RAG2 for origins 8 of antigen-specific immunity
Alka Agrawal*, Quinn M. Eastman† & David G. Schatz‡
* Department of Pharmacology, † Department of Molecular Biophysics and Biochemistry, and ‡ Howard Hughes Medical Institute, Section of Immunobiology, Yale University School of Medicine, New Haven,Connecticut 06510, USA
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Immunoglobulin and T-cell-receptor genes are assembled from component gene segments in developing lymphocytesby 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 containingrecombination 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 splitnature 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.

Lymphocytes of the vertebrate adaptive system rely on a diverse array of immunoglobulins and T-cell antigen receptors (TCRs) for specific recognition of antigens. In the germ line, the genesencoding the variable portions of these receptors are typically split into component V (variable), J (joining) and, in some cases, D (diversity) gene segments. One of each type of gene segment is joined together in a site-specific recombination reaction to form the exon that encodes the antigen-binding portion of the polypeptide1. This reaction, known as V(D)J recombination, occurs only in lymphocytes,and in some vertebrate species is responsible for generating much of the diversity seen in antigen receptors. V, D and J gene segments are flanked by recombination signals that consist of conserved heptamer and nonamer sequences separated by a poorly conserved spacer sequence whose length is usually 12 or 23 base pairs (bp) (referred to hereafter as 12-signals and 23-signals, respectively). Thesignals target the reaction, and efficient recombination only occurs between a 12-signal and a 23-signal, a restriction referred to as the 12/23 rule2. The first phase of the recombination reaction is achieved by the products of the recombination-activating genes, RAG1 and RAG2 (refs 3, 4). Together, the RAG1 and RAG2 proteins bind two recombination signals, bring them into close juxtaposition (thisprocess is termed synapsis), and cleave the DNA, thereby separating the signals from the flanking coding segments5–7. The DNAbending high-mobility group proteins HMG1 and HMG2 substantially enhance the efficiency of coordinate cleavage8,9, in part by improving binding to the 23-signal9. Cleavage occurs in two steps, with a nick first introduced adjacent to the heptamer to expose a 3 -hydroxyl group onthe coding flank, followed by direct nucleophilic attack of the 3 -hydroxyl on the opposite DNA strand5,10. The products are blunt, 5 -phosphorylated signal ends and covalently sealed hairpin coding ends (Fig. 1a). In the second phase of the reaction, the two signal ends are joined precisely to form a signal joint, whereas the coding ends are processed to form a coding joint that typically...