Abstract B073: Structural basis of the 12-23 rule in V(D)J recombination

The V, D and J segment recombination (V(D)J recombination) is the key event in the development and maturation of T and B lymphocytes, which generates a diverse repertoire of T and B cell clones for the further selection of useful specificities. V(D)J recombination is also the basis for antibody diversity. The initiation step of V(D)J recombination is carried out by a pair of lymphocyte specific enzymes, which are called recombination-activating gene 1 and 2 (RAG1 and RAG2). RAG1 and RAG2 associate with each other and with two recognition signal sequences (RSS, 12RSS and 23RSS) adjacent to the coding ends of the V, D and J segments to catalyze the double stranded breaks required for joining. The reaction goes through several catalytic steps that result in formation of the single recombination complex (SC), the paired complex (PC), the cleaved signal complex (CSC) as well as the signal end complex (SEC). In order to understand the mechanisms of how RAG complex functions on DNA, we solved the structures PC and CSC by cryo-electron microscopy near the atomic resolution. Compared with the Apo-RAG complex that constitutes an open conformation, the PC and CSC reveal a closed conformation with RAG1 in one protomer interacts with RAG2 in another protomer. Furthermore, the first nucleotide of the heptamer in the non-transfer strand is flipped out to avoid the steric hindrance of the 39-OH to attack the phosphodiester bond in the transfer strand. Upon nucleophilic attack and hairpin formation, the nucleotide adjacent to the 59-position of the attacked phosphate is also flipped out to accommodate the tensile force in the hairpin DNA. In addition, we found that the conformation changes in the nonamer-binding domain (NBD), which is distorted toward to the 12RSS, and the distorsion requires two turns for the nonamer DNA in the 23RSS to reach the NBD. Finally, we found that two HMGB1 molecules, rather than one, are required to bind and bend both 12RSS and 23RSS DNA to adjust the conformational change of NBD on each side. In summary, our structural models not only uncover the mechanisms of how RAG protein catalyzes the hairpin formation, but also provide the structural basis of the 12-23 rule in V(D)J recombination. Citation Format: Heng Ru, Melissa G. Chambers, Maofu Liao, Hao Wu. Structural basis of the 12-23 rule in V(D)J recombination. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B073.