A hallmark of vertebrate immunity is the diverse repertoire of antigen-receptor genes that results from combinatorial splicing of gene coding segments by V(D)J recombination. The dimeric (RAG1-RAG2)2 recombinase (RAG) recognizes specific recombination signal sequences (RSSs), each containing a heptamer, a spacer of 12 or 23 base pairs, and a nonamer (12-RSS or 23-RSS). RAG only combines one 12-RSS and one 23-RSS, a dogma known as the 12/23 rule that governs the fidelity of V(D)J recombination. RAG introduces precise breaks at RSS-coding segment junctions to generate cleaved RSSs and hairpin coding segments. Here we report cryo-electron microscopy structures of multiple RAG complexes reconstituted with 12-RSS and 23-RSS intermediates and HMGB1 at up to 3.4 angstrom resolution. These structures reveal a closed conformation of RAG upon RSS synapsis, with base flipping and base-specific recognition of RSSs. Distortion at the RSS-coding segment junction and base flipping in the coding segment uncovers the geometry for a two-metal-ion catalytic mechanism. Both 12-RSS and 23-RSS are exceedingly bent by the bound HMGB1. The nonamer-binding domain dimer of RAG1 is flexibly attached to the active site dimer of RAG and is tilted towards the bent 12-RSS but away from the bent 23-RSS in the synaptic complexes, which provides an induced fit mechanism for the 12/23 rule. Collectively, our structures illustrate the elegant mechanisms of RAG-catalyzed V(D)J recombination at the molecular level.

Citation Format: Heng Ru, Melissa G. Chambers, Tian-Min Fu, Alexander B. Tong, Maofu Liao, Hao Wu. Molecular mechanism of V(D)J recombination from synaptic RAG1-RAG2 complex structures [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B122.