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Structure of the RAG1 nonamer binding domain with DNA reveals a dimer that mediates DNA synapsis

Nature Structural & Molecular Biology volume 16, pages 499–508 (2009)Cite this article

Abstract

The products of recombination-activating genes RAG1 and RAG2 mediate the assembly of antigen receptor genes during lymphocyte development in a process known as V(D)J recombination. Lack of structural information for the RAG proteins has hindered mechanistic studies of this reaction. We report here the crystal structure of an essential DNA binding domain of the RAG1 catalytic core bound to its nonamer DNA recognition motif. The RAG1 nonamer binding domain (NBD) forms a tightly interwoven dimer that binds and synapses two nonamer elements, with each NBD making contact with both DNA molecules. Biochemical and biophysical experiments confirm that the two nonamers are in close proximity in the RAG1/2–DNA synaptic complex and demonstrate the functional importance of the protein-DNA contacts revealed in the structure. These findings reveal a previously unsuspected function for the NBD in DNA synapsis and have implications for the regulation of DNA binding and cleavage by RAG1 and RAG2.

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Figure 1: Schematic of RAG-mediated DNA binding and cleavage.
Figure 2: Electron density maps of DNA in the native and 56DNA crystals.
Figure 3: Structure of the RAG1 NBD-DNA complex.
Figure 4: Important protein-DNA and protein-protein contacts in the NBD-DNA crystal structure.
Figure 5: NBD-DNA contacts are important for DNA binding and hairpin formation.
Figure 6: Detection of nonamer synapsis using FRET.
Figure 7: FRET analysis of the DNA and protein requirements for nonamer synapsis.
Figure 8: Structural comparisons of the RAG1 NBD with the DBDs of Hin and Hermes.

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Acknowledgements

We thank the staff at Advanced Photon Source beamline 24ID and National Synchrotron Light Source beamline X25. We are grateful to N. Grindley, L. Regan and A. Miranker for the use of their spectrofluorometers. We also thank W. Eliason and J. Kavran of the Steitz laboratory, S. Unniraman of the Schatz laboratory and the staff of the Center for Structural Biology Core Facility at Yale for their help. This work was supported by the US National Institutes of Health grant AI32524 to D.G.S., training grant T32 GM08283 and a Gershon Fellowship to F.F.Y. This work was also funded by the Howard Hughes Medical Institute (to D.G.S. and T.A.S.).

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  1. Scott Bailey & Satwik Kamtekar

    Present address: Present addresses: Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA (S.B.); Pfizer, Inc., Chesterfield, Missouri, USA (S.K.).,

Authors and Affiliations

  1. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA

    Fang Fang Yin, Scott Bailey, C Axel Innis, Satwik Kamtekar & Thomas A Steitz

  2. Howard Hughes Medical Institute, Yale University, New Haven, Connecticut, USA

    Scott Bailey, C Axel Innis, Thomas A Steitz & David G Schatz

  3. Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut, USA

    Mihai Ciubotaru & David G Schatz

  4. Department of Chemistry, Yale University, New Haven, Connecticut, USA

    Thomas A Steitz

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Contributions

F.F.Y. performed all of the experiments shown; F.F.Y. crystallized the complex and solved the structure with help from S.B., S.K. and C.A.I.; S.B. contributed to the structure refinement and making of Figure 2; all other figures were generated by F.F.Y.; M.C. contributed to the design and interpretation of FRET assays; T.A.S. contributed to experimental design and analysis of the structure; D.G.S. contributed to experimental design and data interpretation; F.F.Y. and D.G.S. wrote the manuscript, which all authors commented on.

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Correspondence to David G Schatz.

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Yin, F., Bailey, S., Innis, C. et al. Structure of the RAG1 nonamer binding domain with DNA reveals a dimer that mediates DNA synapsis. Nat Struct Mol Biol 16, 499–508 (2009). https://doi.org/10.1038/nsmb.1593

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