| During V(D)J recombination, the RAG1 and RAG2 proteins act cooperatively to catalyze a series of DNA bond breakage and strand transfer reactions. Using protein secondary structure prediction algorithms, we have identified a region of RAG1 with possible structural similarities to the active site regions of transposases and retroviral integrases. Based on this information, we have identified two aspartic acid residues in murine RAG1, D600 and D708, that function specifically in catalysis. The results support a model in which RAG1 contains a single, divalent metal cation binding active site that is structurally related to the active sites of bacterial transposases and retroviral integrases and is responsible for all known catalytic functions of the RAG protein complex.; Secondary structure prediction algorithms and other sequence analyses have suggested that RAG2 contains six kelch repeat motifs that are predicted to form a six bladed β-propeller structure, with the second β-strand of each repeat demonstrating marked conservation both within and between kelch repeat-containing proteins. Using a panel of site-directed mutations within the predicted kelch motifs of RAG2, we show that regions within the second β-strand of each kelch repeat are critical for RAG1-RAG2 interaction, recombination signal recognition, and cleavage. We also show that several mutations in this second β-strand are the cause of immunodeficiency in several patients suffering from B−/T− SCID and Omenn Syndrome. Most proteins that contain kelch-like β-propellers are thought to function as molecular scaffolds for multi-protein complexes. This suggests RAG2 may function as an adapter protein that is required for proper RAG1 function. |
