In responding to numerous foreign antigens, the immune system has utilized a clever approach known as V(D)J recombination to generate an enormous antigen receptor repertoire from a small set of genes. V(D)J recombination enables the rearrangement of V(variable), D(diversity), J (joining) gene segments of immunoglobulin and T cell receptor loci by a protein complex composed of RAG-1 and -2. Failure to execute V(D)J recombination leads to severe combined immune deficiency, due to an early block in B and T cell maturation. To cleave DNA at antigen receptor gene loci, the RAG complex first recognizes the flanking recombination signal sequence and generates a nick on one strand, followed by a transesterification reaction that forms one hairpin end and one blunt end. The ends are then joined with the help of DNA repair factors to achieve the recombination of V, D, and J segments.

My graduate work is focused on understanding the molecular mechanism by which RAG generates a DNA hairpin structure during cleavage through identifying amino acid residues in RAG1 that are crucial for this reaction. My work has directly contributed to the identification of a novel motif in RAG1 that may potentially function in a similar way as some hairpin-forming transposases. Transposition, another type of transesterification reaction, can also be carried out by the RAG recombinase. We examined the transposing ability of RAG mutants defective for forming hairpins and their responses to distorted DNA in these two types of transesterfication reactions. We found that RAG mutants severely defective in hairpin formation can still actively transpose, suggesting that these two reactions catalyzed by the RAG complex are not mechanistically identical. Finally, I studied a RAG1 mutant found in patients suffering from Omenn syndrome, an immune deficiency caused by hypomorphic RAG mutant proteins, and demonstrated that it has a conditional defect in forming DNA hairpins during cleavage in vitro, depending on the sequence at the end of coding segment of antigen receptor loci. This preference in selecting gene segments to recombine may provide a novel mechanism to explain the restricted immune repertoire in some Omenn syndrome patients.