Separation-of-function mutants reveal critical roles for RAG2 in both the cleavage and joining steps of V(D)J recombination

The only established physiological function of the V(D)J recombinase, compr ising RAG1 and RAG2, is to perform DNA cleavage. The molecular roles of RAG 2 in cleavage, the mechanisms used to join the broken DNA ends, and the ide ntity of nuclease(s) that open the hairpin coding ends have been unknown. S ite-directed mutagenesis targeting each conserved basic amino acid in RAG2 revealed several separation-of-function mutants that address these question s. Analysis of these mutants reveals that RAG2 helps recognize or cleave di storted DNA intermediates and plays an essential role in the joining step o f V(D)J recombination. Moreover, the discovery that some mutants block RAG- mediated hairpin opening in vitro provides a critical link between this bio chemical activity and coding joint formation in vivo.