RAG-1-DEPENDENT AND RAG-2-DEPENDENT ASSEMBLY OF FUNCTIONAL COMPLEXES WITH V(D)J RECOMBINATION SUBSTRATES IN SOLUTION

V(D)J recombination is initiated by RAG-1 and RAG-2, which introduce d ouble-strand DNA breaks at recombination signal sequences (RSSs) of an tigen receptor gene segments to produce signal ends, terminating in bl unt, double-strand breaks, and coding ends, terminating in DNA hairpin s. While the formation of RAG-RSS complexes has been documented, obser vations regarding the individual contributions of RAG-1 and RAG-2 to R SS recognition are in conflict. Here we describe an assay for formatio n and maintenance of functional RAG-RSS complexes in the course of the DNA cleavage reaction. Under conditions of in vitro cleavage, the RAG proteins sequester intact substrate DNA in a stable complex which is formed prior to strand scission. The cleavage reaction subsequently pr oceeds through nicking and hairpin formation without dissociation of s ubstrate. Notably, the presence of both RAG-1 and RAG-2 is essential f or formation of stable, functional complexes with substrate DNA under conditions of the sequestration assay. Two classes of substrate mutati on are distinguished by their effects on RAG-mediated DNA cleavage in vitro. A mutation of the first class, residing within the RSS nonamer and associated with coordinate impairment of nicking and hairpin forma tion, greatly reduces the stability of RAG association with intact sub strate DNA. In contrast, a mutation of the second class, lying within the RSS heptamer and associated with selective abolition of hairpin fo rmation, has little or no effect on the half-life of the RAG-substrate complex.