title: Autoinhibition of DNA cleavage mediated by RAG1 and RAG2 is overcome by an epigenetic signal in V(D)J recombination.
abstract: Gene assembly of the variable domain of antigen receptors is initiated by DNA cleavage by the RAG1-RAG2 protein complex at sites flanking V, D, and J gene segments. Double-strand breaks are produced via a single-strand nick that is converted to a hairpin end on coding DNA and a blunt end on the neighboring recombination signal sequence. We demonstrate that the C-terminal regions of purified murine RAG1 (aa 1009-1040) and RAG2 (aa 388-520, including a plant homeodomain [PHD domain]) collaborate to inhibit the hairpinning stage of DNA cleavage. The C-terminal region of RAG2 stabilizes the RAG1/2 heterotetramer but destabilizes the RAG-DNA precleavage complex. This destabilization is reversed by binding of the PHD domain to a histone H3 peptide trimethylated on lysine 4 (H3K4me3). The addition of H3K4me3 likewise alleviates the RAG1/RAG2 C-terminus-mediated inhibition of hairpinning and the PHD-mediated inhibition of transposition activity. Thus a negative regulatory function of the noncore regions of RAG1/2 limits the RAG endonuclease activity in the absence of an activating methylated histone tail bound to the complex.
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Gabrielle J
authors.initials: GJ
authors.lastname: Grundy
authors.forename: Wei
authors.initials: W
authors.lastname: Yang
authors.forename: Martin
authors.initials: M
authors.lastname: Gellert
mesh_terms: D000818:Animals; D001665:Binding Sites; D053837:DNA Cleavage; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D057890:Epigenomics; D057809:HEK293 Cells; D006657:Histones / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008745:Methylation; D051379:Mice; D009154:Mutation; D011485:Protein Binding; D055503:Protein Multimerization; D011995:Recombination, Genetic; D015398:Signal Transduction; D050441:VDJ Exons / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D006657:Histones; D018398:Homeodomain Proteins; C499308:Rag2 protein, mouse; C064658:RAG-1 protein; D008239:Lysine