title: Initiation of V(D)J recombination in vitro obeying the 12/23 rule.
abstract: V(D)J recombination, the process that assembles antigen-receptor genes, is directed by signal sequences flanking the DNA segments to be joined. Signals consist of a conserved heptamer and nonamer separated by a spacer of either 12 or 23 base pairs. Recombination occurs almost exclusively between two signals with spacers of different lengths. This restriction, called the '12/23 rule', governs the organization and pattern of rearrangement of antigen-receptor loci. In vitro work demonstrating the direct roles of the Rag proteins in the initiation of V(D)J recombination did not recreate the 12/23 rule. Instead, double-strand breaks were formed efficiently at isolated signals. Here we show that extracts made from a lymphoid cell line that expresses truncated forms of the Rag1 and Rag2 proteins have a signal-cutting activity that obeys the 12/23 rule. Cleavage at the two signals is concerted and requires their synapsis, and mutations of one signal prevent cleavage at both.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520-8011, USA.
authors.forename: Q M
authors.initials: QM
authors.lastname: Eastman
authors.forename: T M
authors.initials: TM
authors.lastname: Leu
authors.forename: D G
authors.initials: DG
authors.lastname: Schatz
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011946:Receptors, Antigen / Q000235:genetics*; D011995:Recombination, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D014162:Transfection
chemical_list: D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D011506:Proteins; D011946:Receptors, Antigen; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004247:DNA