Adaptive immunity is the highest form of specific immunity in jawed vertebrates. Its power is predominantly due to the large and diverse repertoire of antigen receptor genes including antibodies as well as T and B cell receptors for both humoral and cell-mediated immune response during pathogen invasion and tumor surveillance. The diversity of antibody and T and B cell receptors are generated through the combinatorial splicing of V, D and J gene segments in the variable region of antigen receptor genes at the DNA level during lymphocyte development. The V(D)J recombination is initiated by the dimeric (RAG1-RAG2)2 complex which specifically recognizes a pair of recombination signal sequences, combinatorially pairs gene segments, and presents hairpin gene segments for opening and ligation by DNA damage repair machineries. An important dogma for RAG functions has been the so-called 12/23 rule, which governs the fidelity of the recombination process. In this study, Ru and his colleagues solved the cryo-EM structures of synaptic RAG in complex with various forms of DNA intermediates and products, at near-atomic resolutions. The structures of the synaptic RAG complexes reveal a closed dimer conformation with generation of new intermolecular interactions between two RAG1-RAG2 monomers upon DNA binding, compared to the Apo-RAG complex which constitutes as an open conformation. Both RAG1 molecules in the closed dimer are involved in the cooperative binding of the 12-RSS and 23-RSS intermediates with base specific interactions in the heptamer of the signal end. The first base of the heptamer in the signal end is flipped out to avoid the clash in the active center. Each coding end of the nicked-RSS intermediate is stabilized exclusively by one RAG1-RAG2 monomer with non-specific protein-DNA interactions. The coding end is highly distorted with one base flipped out from the DNA duplex in the active center, which facilitates the hairpin formation by a potential two-metal ion catalytic mechanism. The 12-RSS and 23-RSS intermediates are highly bent and asymmetrically bound to the synaptic RAG complex with the nonamer binding domain dimer tilts towards the nonamer of the 12-RSS but away from the nonamer of the 23-RSS, which emphasizes the 12/23 rule. Two HMGB1 molecules bind at each side of 12-RSS and 23-RSS to stabilize the highly bent RSSs. These structures elaborate the molecular mechanisms for DNA recognition, catalysis and the unique synapsis underlying the 12/23 rule, provide new insights into the RAG-associated human diseases, and represent a most complete set of complexes in the catalytic pathways of any DDE family recombinases, transposases or integrases.
Ru H1, Chambers MG2, Fu TM1, Tong AB1, Liao M3, Wu H4.[expand title=”Show Affiliations”]
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115, USA.
- Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
- Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. Electronic address: [email protected]
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115, USA. Electronic address: [email protected] [/expand]
Diverse repertoires of antigen-receptor genes that result from combinatorial splicing of coding segments by V(D)J recombination are hallmarks of vertebrate immunity. The (RAG1-RAG2)2 recombinase (RAG) recognizes recombination signal sequences (RSSs) containing a heptamer, a spacer of 12 or 23 base pairs, and a nonamer (12-RSS or 23-RSS) and introduces precise breaks at RSS-coding segment junctions. RAG forms synaptic complexes only with one 12-RSS and one 23-RSS, a dogma known as the 12/23 rule that governs the recombination fidelity. We report cryo-electron microscopy structures of synaptic RAG complexes at up to 3.4 Å resolution, which reveal a closed conformation with base flipping and base-specific recognition of RSSs. Distortion at RSS-coding segment junctions and base flipping in coding segments uncover the two-metal-ion catalytic mechanism. Induced asymmetry involving tilting of the nonamer-binding domain dimer of RAG1 upon binding of HMGB1-bent 12-RSS or 23-RSS underlies the molecular mechanism for the 12/23 rule.
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