Structural and mechanistic studies of V(D)J recombination

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

V(D)J recombination is the site-specific DNA rearrangement that assembles antigen receptor genes from dispersed arrays of V, D and J gene segments. Recombination is initiated by the lymphoid-specific RAG1 and RAG2 recombinase, which recognizes and cleaves the recombination signal sequences. V(D)J recombination is tightly regulated, occurring in a preferred temporal order and only in specific cell types and developmental stages. Immunoglobulin (Ig) heavy chain rearrangement precedes light chain rearrangement and Ig heavy-chain D to J joining precedes V to DJ recombination. In addition, Ig genes are fully rearranged only in B cells (not T cells) and T cell receptor genes are assembled in T but not B cells. Over-expression of RAG1 and RAG2 in non-lymphoid cells is sufficient to induce recombination of an artificial extrachromosomal substrate, but does not support V(D)J recombination of endogenous loci. Therefore, the accessibility of these loci to the recombinase must be regulated. A large body of evidence suggests that the regulation of chromatin structure is involved in the regulation of V(D)J recombination. We have finally succeeded in determining the structures of RAG1/RAG2 recombinase and their complex with recombination-signal sequence (RSS) in the process of DNA cleavage. We showed the series of conformational changes of RAG1/2 during the first and second strand of DNA cleavage. We also used structural biology to elucidate why RAG1/2 is a recombinase and not a transposase. In collaboration with Dr. Martin Gellerts lab, in 2020 we published two paper on cryoEM structures of RAG1/2 as a DNA recombinase. These papers conclude the first phase of studying V(D)J recombination. In addition, we published cryoEM structures DNA-PK, which is the central organizer of the non-homologous end-joining process. For the DNA hairpin opening by Artemis, we collaborated with Drs. Marty Gellert and Kathy Meeks, the results are published in two Molecular Cell papers since 2021. In 2022-23, we have made progresses in capturing the DNA filling and ligation complexes in NHEJ. In In 2023-24, we have refined the structures of gap filling by DNA pol mu and end-joining by Ligase IV (LIG4). The results are written up and the manuscript is under review. in 2024-25, we have completed the analyses of DNA gap-filling and DNA-end joining reactions in NHEJ and elucidated the molecular mechanism for the dynamic assemblies and coordinated reactions that complete the NHEJ process. References: Liu, L., Li, J., Cisneros-Aguirre, M., Merkell, A., Stark, J.M., Gellert, M. & Yang, W. (2025) Dynamic assemblies and coordinated reactions of non-homologous end joining, Nature, doi: 10.1038/s41586-025-09078-9. Liu, L., Chen, X.M., Li, J., Wang, H., Buehl, C.J., Goff, N.J., Meek, K., Yang, W. & Gellert, M. (2021) Autophosphorylation transforms DNA-PK from protecting to processing of DNA ends. Mol Cell, 82(1):177-189 (ePub on 12/21/2021). Chen, X., Xu, X., Chen, Y., Cheung, J.C., Wang, H., Jiang, J., de Val, N., Fox, T., Gellert, M. & Yang, W. (2021) Structure of an activated DNA-PK and its implications for NHEJ. Mol Cell, 81, 801-810. Chen, X.M., Cui, Y.X., Wang, H., Zhou, Z.H., Gellert, M. & Yang, W. (2020) How RAG1/2 recombinase avoids DNA transposition. NSMB, 27, 127-133. Chen, X.M., Cui, Y.X., Best, R.B., Wang, H., Zhou, Z.H., Yang, W. & Gellert, M. (2020) Cutting antiparallel DNA strands in a single active site. NSMB, 27, 119-126. Kim, M.S., Lapkouski, M., Yang, W. & Gellert M. (2015) Crystal structure of the V(D)J recombinase RAG1-RAG2, Nature, 518, 507-511.