Identification of basic residues in RAG2 critical for DNA binding by the RAG1-RAG2 complex

In V(D)J recombination, the RAG1 and RAG2 proteins are the essential compon ents of the complex that catalyzes DNA cleavage. RAG1 has been shown to pla y a central role in DNA binding and catalysis. In contrast, the molecular r oles of RAG2 in V(D)J recombination are unknown. To address this, we indivi dually mutated 36 evolutionarily conserved basic and hydroxy group containi ng residues within RAG2. Biochemical analysis of the recombinant RAG2 prote ins led to the identification of a number of basic residue mutants defectiv e in catalysis in vitro and V(D)J recombination in vivo. Five of these were deficient in binding of the RAG1-RAG2 complex to its cognate DNA target se quence while interacting normally with RAG1. Our findings provide support f or the direct involvement of RAG2 in DNA binding during all steps of the cl eavage reaction.