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A Biochemically Defined System for Coding Joint Formation in V(D)J Recombination
Author(s) -
Lu Haihui,
Schwarz Klaus,
Swanson Patrick C.,
Lieber Michael R.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.2_supplement.415
Subject(s) - v(d)j recombination , recombination , dna , recombination signal sequences , biology , flp frt recombination , non homologous end joining , gene , recombination activating gene , site specific recombination , microbiology and biotechnology , genetics , genetic recombination , recombinase
Evolved from invertebrate transposons, V(D)J recombination is the vertebrate gene rearrangement process for assembling the antigen receptor genes encoding immunoglobulins and T cell receptors. It is also one of the most complex DNA transactions in biology. The RAG complex initiates V(D)J recombination by making double‐strand breaks adjacent to signal sequences and creating hairpin coding ends. Here we find that the kinase and endonuclease activity of the DNA‐PKcs:Artemis complex can be activated by hairpin DNA ends, thereby allowing the hairpins to be opened and then to undergo processing and joining by nonhomologous DNA end joining. Based on these insights, we have reconstituted many aspects of the antigen receptor diversification of V(D)J recombination using 13 highly purified proteins, thereby permitting variable domain exon assembly using this fully defined system. The features of the recombination sites created by this system include all of the characteristics observed in vivo (nucleolytic resection, P nucleotides, and N nucleotide addition), indictating that most, if not all, of the end modification enzymes have been identified. This in vitro system provides a novel tool for detailed mechanistic analysis of V(D)J recombination.