The RAG1 and RAG2 proteins establish the 12/23 rule in V(D)J recombination

V(D)J recombination requires a pair of signal sequences with spacer lengths of 12 and 23 base pairs. Cleavage by the RAG1 and RAG2 proteins was previously shown to demand only a single signal sequence. Here, we establish conditions where 12- and 23-spacer signal sequences are both necessary for cleavage. Coupled cuffing at both sites requires only the RAG1 and RAG2 proteins, but depends on the metal ion. In Mn²⁺, a single signal sequence supports efficient double strand cleavage, but cuffing in Mg²⁺ requires two signal sequences and is best with the canonical 12/23 pair. Thus, the RAG proteins determine both aspects of the specificity of V(D)J recombination, the recognition of a single signal sequence and the correct 12/23 coupling in a pair of signals.