Mechanistic basis for coding end sequence effects in the initiation of V(D)J recombination

V(D)J recombination is directed by recombination signal sequences. However, the flanking coding end sequence can markedly affect the frequency of the initiation of V(D)J recombination in vivo. Here we demonstrate that the cod ing end sequence effect can be qualitatively and quantitatively recapitulat ed in vitro with purified RAG proteins. We find that coding end sequence sp ecifically affects the nicking step, which is the first biochemical step in RAG-mediated cleavage. The subsequent hairpin formation step is not affect ed by the coding end sequence. Furthermore,the coding end sequence effect c an be ablated by prenicking the substrate, indicating that the coding end e ffect is specific to the nicking step. In reactions in which both 12- and 2 3-substrates are present, a suboptimal coding end sequence on one signal ca n slow down hairpin formation at the partner signal, a result consistent wi th models in which coordination between the signals occurs at the hairpin f ormation step. The coding end sequence effect an nicking and the coupling o f the 12- and 23-substrates explains how hairpin formation can be rate limi ting for some 12/23 pairs, whereas nicking can be rate limiting when low-ef ficiency coding end sequences are involved.