RAG2’s Acidic Hinge Restricts Repair-Pathway Choice and Promotes Genomic Stability

• The acidic hinge in RAG2 prevents aberrant repair of RAG-mediated DNA breaks
• Acidic residues strengthen the RAG/DNA postcleavage complex and enhance flexibility
• Acidic residues preserve genomic stability in lymphocytes during V(D)J recombination
• Experiments with identified sequence variants suggest similar mechanism in humans

SummaryV(D)J recombination-associated DNA double-strand breaks (DSBs) are normally repaired by the high-fidelity classical nonhomologous end-joining (cNHEJ) machinery. Previous studies implicated the recombination-activating gene (RAG)/DNA postcleavage complex (PCC) in regulating pathway choice by preventing access to inappropriate repair mechanisms such as homologous recombination (HR) and alternative NHEJ (aNHEJ). Here, we report that RAG2’s “acidic hinge,” previously of unknown function, is critical for several key steps. Mutations that reduce the hinge’s negative charge destabilize the PCC, disrupt pathway choice, permit repair of RAG-mediated DSBs by the translocation-prone aNHEJ machinery, and reduce genomic stability in developing lymphocytes. Structural predictions and experimental results support our hypothesis that reduced flexibility of the hinge underlies these outcomes. Furthermore, sequence variants present in the human population reduce the hinge’s negative charge, permit aNHEJ, and diminish genomic integrity.

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