RAG1 mutations affect cutting and binding in vitro. (A) Cutting at a 12-RSS (left) or 23-RSS (right) is shown. An oligonucleotide carrying a consensus 12- or 23-RSS, as indicated, was incubated with equivalent amounts of core RAG2 (cRAG2) plus the core RAG1 (cRAG1) proteins shown (C) in the presence of HMGB1, with a 10-fold excess of unlabeled 23- or 12- partner RSS. For cutting reactions on the far right of each gel, equal amounts of R401W and R504Q were mixed to give the same total amount of RAG1 as the other lanes. The percentage cutting is indicated beneath the gels; an asterisk indicates the labeled oligonucleotide. (B) Binding to a labeled 12-RSS (left) and 23-RSS (right). Binding reactions were performed with cRAG2 and the cRAG1 proteins indicated. Complexes SC1 and SC2, as well as HSC1 and HSC2, are shown. (C) The levels of purified RAG proteins are equivalent. Purified, maltose binding protein-tagged cRAG1 and cRAG2 proteins were separated by electrophoresis and the gel stained with Coomassie blue. The various RAG1 proteins are indicated above each lane. (D) Comparison of R401W and WT RAG1 binding to a 12-RSS. RAG binding reactions were performed in the presence or absence of HMGB1, as indicated. Whereas R401W forms equivalent complexes to WT with the 12-RSS, in the absence of HMGB1, R401W does not form HSC1.