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Molecular Mechanism of V(D)J Recombination from Synaptic RAG1-RAG2 Complex Structures

Significance Statement

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.

About The Author

Dr. Heng Ru obtained his Ph.D. in Biochemistry and Molecular Biology from the Institute of Biophysics, Chinese Academy of Sciences in 2012. During his Ph.D. training, he mainly focused on mechanistic understanding of innate immune responses against pathogen infections, especially on the molecular mechanism of DNA recognition by cytosolic double-stranded DNA (dsDNA) sensors as well as its downstream signaling. He determined several structures of HIN domains of AIM2-like receptors (ALRs) including AIM2, Ifi16 and p202, and their complexes with dsDNA, as well as the adaptor molecule STING in the apo-form and the pathogenic agonist, c-di-GMP cyclic dinucleotide bound state, by X-ray crystallography. He then joined Dr. Hao Wu’s Lab as a postdoc research fellow at Program in Cellular and Molecular Medicine, Boston Children’s Hospital, and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School after his graduation. His research focuses on the molecular mechanism and structural basis of RAG1-RAG2 complex in the initial stage of V(D)J recombination.  

About The Author

Dr. Hao Wu received her pre-medical training at Peking University from 1982-1985 and studied Medicine at Peking Union Medical College from 1995-1988. She obtained her Ph.D. from Purdue University in 1992 and performed postdoctoral research at Columbia University. She became an Assistant Professor at Weill Cornell Medical College in 1997 and was promoted to Professor in 2003. In 2012, Dr. Wu moved to Harvard Medical School as the Asa and Patricia Springer Professor of Biological Chemistry and Molecular Pharmacology, and Boston Children’s Hospital as Senior Investigator in the Program in Cellular and Molecular Medicine. Dr. Wu has received a number of honors, including the Pew Scholar award, the Rita Allen Scholar award, the Margaret Dayhoff Memorial Award from the Biophysical Society. She is an elected member of the National Academy of Sciences.  

About The Author

Dr. Maofu Liao received his Bachelor of Science degree in 1999 in Tsinghua University. He joined the Biomedical Science graduate program in Albert Einstein College of Medicine, worked in Dr. Margaret Kielian’s laboratory to study the mechanism of membrane fusion mediated by viral proteins, and obtined his Ph.D. degree in 2006. He then joined Dr. Yifan Cheng’s laboratory at UCSF in 2008 to study protein structure and fuction using cryo-electron microscopy (cryo-EM). Taking the advantage of the newest technologies in single-particle cryo-EM, he determined the near-atomic resolution structures of the TRPV1 ion channel, without the need of forming protein crystals. In 2014, he became an assistant professor in the Department of Cell Biology at Harvard Medical School. The research interests of his laboratory focus on understanding the structure and function of membrane proteins and protein-DNA complexes, and he mainly uses single-particle cryo-EM and a variety of biochemical assays to reveal the underlying mechanisms of these molecular machines in their native environments.

Molecular Mechanism of V(D)J Recombination from Synaptic RAG1-RAG2 Complex Structures.

 

Journal Reference

Cell. 2015;163(5):1138-52.

Ru H1, Chambers MG2, Fu TM1, Tong AB1, Liao M3, Wu H4.

Show Affiliations
  1. 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.
  2. Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
  3. Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. Electronic address: [email protected]
  4. 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]

Abstract

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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