Detection of RAG protein-V(D)J recombination signal interactions near the site of DNA cleavage by UV cross-linking

V(D)J recombination is initiated by double-strand cleavage at recombination signal sequences (RSSs). DNA cleavage is mediated by the RAG1 and RAG2 pro teins. Recent experiments describing RAG protein-RSS complexes, while defin ing the interaction of RAG1 with the nonamer, have not assigned contacts im mediately adjacent to the site of DNA cleavage to either RAG polypeptide. H ere we use UV cross-linking to define sequence- and site-specific interacti ons between RAG1 protein and both the heptamer element of the RSS and the c oding Bank DNA. Hence, RAG1-DNA contacts span the site of cleavage. We also detect cross-linking of RAG2 protein to some of the same nucleotides that cross-link to RAG1, indicating that, in the binding complex, both RAG prote ins are in close proximity to the site of cleavage. These results suggest h ow the heptamer element, the recognition surface essential for DNA cleavage , is recognized by the RAG proteins and have implications for the stoichiom etry and active site organization of the RAG1-RAG2-RSS complex.