Novel insights into the biochemical mechanisms of RAG1 and RAG2 using mutational approaches

V(D)J recombination generates a diverse repertoire of antigen receptors by somatically recombining different gene segments. The reaction is catalysed by the proteins RAG1 and RAG2 which bind and cleave recombination signal sequences flanking each gene segment. However, V(D)J recombination is a daring approach since it involves the breaking and rejoining of the genome, posing a threat to genome stability. Indeed, mistakes do occur, highlighted by 35-40% of lymphoid cancers bearing hallmarks of errors during antigen receptor production. Additionally, mutation of the RAG proteins can cause immunodeficiency, the severity of which reflects the importance of the altered residue(s) during recombination. Here, I study the RAG proteins in two different ways: using naturally occurring or artificial mutations. Firstly, I analysed two RAG1 mutations identified in an immunodeficient patient. Both mutations lead to a severe decrease in recombination in vivo, and analysis of the reaction in vitro revealed that one mutation disrupts the recruitment of the accessory protein HMGB1 to the RAG complex. Together with studies of recombination in HMGB1 deficient cells, this revealed a critical in vivo role for HMGB1/2 during V(D)J recombination. Next, I used artificially generated RAG2 mutations in a bespoke in vivo assay to map the region of the RAG2 C-terminus that inhibits an aberrant reaction, namely reintegration, where recombination by-products are reinserted into the genome. This identified four residues in the RAG2 acidic hinge which are vital to suppress this dangerous reaction. Finally, I generated novel chromatin substrates, containing the H3K4me3 modification, to test a model for how the RAG proteins maintain genome integrity. This led to the unexpected discovery that the RAG2 C-terminus inhibits binding and cleavage of nucleosome substrates which lack the H3K4me3 modification. Together, these studies provide novel insights into the function of RAG proteins.