The Recombination Activating Genes, RAG1 and RAG2, are essential for V(D)J recombination and adaptive immunity. Mutations in these genes often cause immunodeficiency, the severity of which reflects the importance of the altered residue or residues during recombination. Here, we describe a novel RAG1 mutation that causes immunodeficiency in an unexpected way: The mutated protein severely disrupts binding of the accessory protein, HMGB1. Although HMGB1 enhances RAG cutting in vitro, its role in vivo was controversial. We show here that reduced HMGB1 binding by the mutant protein dramatically reduces RAG cutting in vitro and almost completely eliminates recombination in vivo. The RAG1 mutation, R401W, places a bulky tryptophan opposite the binding site for HMG Box A at both 12- and 23-spacer recombination signal sequences, disrupting stable binding of HMGB1. Replacement of R401W with leucine and then lysine progressively restores HMGB1 binding, correlating with increased RAG cutting and recombination in vivo. We show further that knockdown of HMGB1 significantly reduces recombination by wild-type RAG1, whereas its re-addition restores recombination with wild-type, but not the mutant, RAG1 protein. Together, these data provide compelling evidence that HMGB1 plays a critical role during V(D)J recombination in vivo.
© 2019 by The American Society of Hematology.