RAG-2 promotes heptamer occupancy by RAG-1 in the assembly of a V(D)J initiation complex

V(D)J recombination occurs at recombination signal sequences (RSSs) contain ing conserved heptamer and nonamer elements. RAG-1 and RAG-2 initiate recom bination by cleaving DNA between heptamers and antigen receptor coding segm ents, RAG-1 alone contacts the nonamer but interacts weakly, if at all, wit h the heptamer. RAG-2 by itself has no DNA-binding activity but promotes he ptamer occupancy in the presence of RAG-1; how RAG-2 collaborates with RAG- 1 has been poorly understood. Here we examine the composition of RAG-RSS co mplexes and the relative contributions of RAG-1 and RAG-2 to heptamer bindi ng. RAG-1 exists as a dimer in complexes with an isolated RSS bearing a 12- bp spacer, regardless of whether RAG-2 is present; only a single subunit of RAG-1, however, participates in nonamer binding. In contrast, multimeric R AG-2 is not detectable by electrophoretic mobility shift assays in complexe s containing both RAG proteins. DNA-protein photo-cross-linking demonstrate s that heptamer contacts, while enhanced by RAG-2, are mediated primarily b y RAG-1. RAG-2 cross-linking, while less efficient than that of RAG-1, is d etectable near the heptamer-coding junction. These observations provide evi dence that RAG-2 alters the conformation or orientation of RAG-1, thereby s tabilizing interactions of RAG-1 with the heptamer, and suggest that both p roteins interact with the RSS near the site of cleavage.