The role of the carboxy terminus of RAG2 in V(D)J recombination

During immune system development, immunoglobulin and T cell receptor genes must be assembled from their component gene segments. This process, called V(D)J recombination, is initiated by the lymphoid specific R&barbelow;ecombination A&barbelow;ctivating G&barbelow;enes 1 and 2 (RAG1 and 2). Purification of the RAG proteins has allowed extensive study of the V(D)J cleavage reaction in vitro; however, most of this work has been performed with catalytically active "core" versions of RAG1 and RAG2 which were more amenable to purification than the full-length versions. In order to investigate the contribution of the "non-essential" regions of RAG1 and RAG2, I purified full-length versions of the proteins and compared their activity to the core proteins in vitro. I found full-length RAG2 is more active in in vitro cleavage than core RAG2, but that full-length RAG2 blocks the transposition of signal ends. In contrast, full-length RAG1 and RAG2 do support hybrid joint formation in vitro.; Although there was no structural data available for RAG2, sequence-based predictions suggested that the C-terminus of RAG2 folds into a PHD domain. Early studies of other proteins containing PHD domains proposed that the motif acted as a protein-protein interaction module. I undertook a yeast two hybrid screen to search for a protein that would bind to the C-terminus of RAG2, but was unable to recover any interacting proteins. However, recent work has demonstrated that the PHD domains from several different proteins interact with phosphoinositides (PtdInsPs). In collaboration with Or Gozani (Junying Yuan's laboratory) and Dmitri Ivanov (Gerhard Wagner's laboratory), we have confirmed that the region does fold into a non-canonical PHD finger and that the PHD finger of RAG2 interacts with PtdInsPs. I examined several mutant derivatives of RAG2 which are impaired in their ability to bind PtdInsPs and found that these derivatives have defects in V(D)J recombination in vivo but not to a significant extent in vitro. Together, these data confirm the designation of the C-terminus of RAG2 as a PHD finger and implicate the interaction with PtdInsPs in a role in the regulation of V(D)J recombination in vivo.