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DNA melting initiates the RAG catalytic pathway

Nature Structural & Molecular Biology volume 25, pages 732–742 (2018)Cite this article

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Abstract

The mechanism for initiating DNA cleavage by DDE-family enzymes, including the RAG endonuclease, which initiates V(D)J recombination, is not well understood. Here we report six cryo-EM structures of zebrafish RAG in complex with one or two intact recombination signal sequences (RSSs), at up to 3.9-Å resolution. Unexpectedly, these structures reveal DNA melting at the heptamer of the RSSs, thus resulting in a corkscrew-like rotation of coding-flank DNA and the positioning of the scissile phosphate in the active site. Substrate binding is associated with dimer opening and a piston-like movement in RAG1, first outward to accommodate unmelted DNA and then inward to wedge melted DNA. These precleavage complexes show limited base-specific contacts of RAG at the conserved terminal CAC/GTG sequence of the heptamer, thus suggesting conservation based on a propensity to unwind. CA and TG overwhelmingly dominate terminal sequences in transposons and retrotransposons, thereby implicating a universal mechanism for DNA melting during the initiation of retroviral integration and DNA transposition.

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Fig. 1: Cryo-EM structure determination.
Fig. 2: Overview of cryo-EM maps and models.
Fig. 3: DNA distortion and active sites in the RNH domains for unmelted, melted, and nicked RSSs.
Fig. 4: Structural comparisons of RAG monomers in different states.
Fig. 5: Opening of the RAG dimer after RSS substrate binding.
Fig. 6: Structural comparisons of RSS recognition and key conserved RSS interactions.
Fig. 7: Conformational transitions in the RAG catalytic pathway and implications for DNA transposases and retroviral integrase of the DDE family.

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Acknowledgements

Some of this work was performed with the assistance of H. Wei at the Simons Electron Microscopy Center and the National Resource for Automated Molecular Microscopy at the New York Structural Biology Center, supported by grants from the Simons Foundation (349247), NYSTAR, and the NIH National Institute of General Medical Sciences (GM103310). This work was supported by the National Cancer Institute’s National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research. We also acknowledge research funding from the National Institutes of Health (AI125535 to H.W. and M.L., AI0240067-36 to F.W.A., and AI032524 to D.G.S.) and an Irvington Postdoctoral Fellowship from the Cancer Research Institute (H.R.). F.W.A. is supported as an HHMI investigator.

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Author notes

  1. These authors contributed equally: Heng Ru, Wei Mi.

Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    Heng Ru, Pengfei Zhang & Hao Wu

  2. Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA, USA

    Heng Ru, Pengfei Zhang, Frederick W. Alt & Hao Wu

  3. Department of Cell Biology, Harvard Medical School, Boston, MA, USA

    Wei Mi & Maofu Liao

  4. Department of Genetics, Harvard Medical School, Boston, MA, USA

    Frederick W. Alt

  5. Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA

    Frederick W. Alt

  6. Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA

    David G. Schatz

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Contributions

H.R., H.W., and F.W.A. conceived and initiated the project. H.W. and H.R. participated in project design and analysis. H.R. prepared RAG complexes for biochemical studies and EM experiments. P.Z. assisted with preliminary biochemical assays. W.M. performed cryo-EM data collection on a Polara microscope at Harvard Medical School, and W.M., H.R., and H.W. participated in cryo-EM data collection on a Titan Krios microscope at the Simons Electron Microscopy Center. M.L., H.R., W.M., and H.W. carried out image processing and reconstruction. H.R. performed model building, refinement, and calculation of FSC curves. H.W., H.R., D.G.S., M.L., and F.W.A. performed data analysis, and discussion and interpretation of results. H.W. and H.R. made the figures and wrote the manuscript, and all authors edited the manuscript.

Corresponding authors

Correspondence to Maofu Liao or Hao Wu.

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Integrated supplementary information

Supplementary Figure 1 V(D)J recombination process and DNA sequences used in this study.

a, Schematic representation of a simplified V(D)J recombination process at the human lgH locus. V, D, J and constant regions are shown as light purple, light green, light blue and gray rectangles, and 12- and 23-RSSs are shown as red and orange triangles. Randomly incorporated nucleotides between V, D and J coding segments are shown as blue rectangles. b, 12-RSS and 23-RSS sequences used in this study. Heptamer sequences (red) are numbered from 1 to 7 for the forward strand and from 1* to 7* for the reverse strand. Coding flank sequences are numbered from −16 to −1 for the forward strand and from −16* to −1* for the reverse strand. The nicking site is between A–1 (coding flank) and C1 (RSS) with a 3’-OH on A–1.

Supplementary Figure 2 Cryo-EM structure determination.

a, A flow chart of cryo-EM structure determination for the data collected using an FEI Titan Krios microscope on an intact DNA complex sample prepared at 21 °C. Final maps are enclosed in black boxes. b, A flow chart of cryo-EM structure determination for the combined data collected using an FEI Polara microscope on an intact DNA complex sample prepared at 37 °C and using an FEI Titan Krios microscope on an intact DNA complex sample prepared at 21 °C. Final maps are enclosed in black boxes. Some major classes were not pursued further because they either did not contain bound DNA or contained a DNA state for which we already had a reconstruction at a good resolution.

Supplementary Figure 3 Cryo-EM structure determination

a, An alternative flow chart of cryo-EM structure determination for the combined data collected using an FEI Polara microscope on an intact DNA complex sample prepared at 37 °C and using an FEI Titan Krios microscope on an intact DNA complex sample prepared at 21 °C. Final maps are enclosed in black boxes. b, A flow chart of cryo-EM structure determination for the RAG in complex with nicked RSS intermediates. Final maps are enclosed in black boxes. Some major classes were not pursued further because they either did not contain bound DNA or contained a DNA state for which we already had a reconstruction at a good resolution.

Supplementary Figure 4 Fourier shell correlation curves for all the maps.

Black lines: FSC curves between one half-map to the other half-map. Red lines: FSC curves between a final refined model and the sum of two half-maps. Green lines: FSCwork curves between a half-map and a model refined against the same half-map. Blue lines: FSCfree curves between a half-map and a model refined against the other half-map. Black and red dashed lines indicate the resolutions at FSC = 0.143 and 0.5 for half maps to half maps and models to maps, respectively.

Supplementary Figure 5 Overview of cryo-EM maps and models.

a,b, Cryo-EM maps of RAG singly bound to unmelted (a) and melted (b) 12-RSS at 4.2 and 4.7 Å resolution. One RAG1 is omitted in order to present the bound 12-RSS DNAs (red) inside the protein. The black circle in (b) indicates the melted region in the RSS. c, Cryo-EM map of C2-symmetrized RAG doubly bound to nicked RSSs at 3.0 Å resolution. One RAG1 is omitted in order to present the bound nicked-RSS DNAs (red and orange) inside the protein. d, Orthogonal view of cryo-EM map of RAG doubly bound to melted 12- and 23-RSS at 5.0 Å resolution. One RAG1 is omitted in order to present the bound RSS DNAs (red and orange) inside the protein. The black circle indicates the melted region in the RSS. e, Ribbon diagram of the RAG model doubly bound to unmelted 12- and 23-RSS at 4.3 Å resolution. RAG1: lemon green and cyan; RAG2: green and slate. 12-RSS and 23-RSS are shown in salmon, except that heptamers are highlighted in red and nonamers in magenta. f, Orthogonal view of C2 symmetrized cryo-EM map of RAG doubly bound to melted RSSs at 4.4 Å resolution. One RAG1 is omitted (left) in order to present the bound RSS DNAs (red and orange) inside the protein. The black circle indicates the melted region in the RSS. g-i, Representative protein regions of the 4.0 Å-resolution averaged map with one RSS melted and the other RSS unmelted (shown in mesh) superimposed with the final models (shown in ribbon). These regions include α22 (I954-E970) and α23 (I975-M996) helices of the first RAG1 molecule for the melted DNA (g), β-sheet region (F611-M622, K638-I651 and F722-K731) of RNH domain of the first RAG1 molecule for the melted DNA (h) and β4-β5 loop region (V625-K638) of the second RAG1 molecule relative to the melted DNA (i). Cryo-EM densities for catalytic residues D620, D730 and E984 as well as the two bound Ca2+ ions in the active site center are indicated. j, Representative DNA regions of the cryo-EM maps (shown in mesh) superimposed to the final models with the phosphate backbones in sticks and bases in cartoons. The regions include positions −4 to +8 of 12-RSS whose sequences are shown in Supplementary Fig. 1b. Left: melted RSS from the 4.0 Å-resolution averaged map with one RSS melted and the other RSS unmelted; middle: unmelted RSS from the 3.9 Å-resolution C2-symmetrized map with unmelted RSSs; right: nicked RSS from the 3.0 Å-resolution C2-symmetrized map with nicked RSSs. Cryo-EM densities for the three nucleotides CAC in the heptamer are indicated.

Supplementary Figure 6 Comparison of the DNAs and proteins at different states.

a,b, Detailed interactions of the RNH domain (yellow) from the 1st RAG1 molecule with unmelted (a) and melted (b) RSS (pale cyan) with heptamer highlighted in salmon. The 1st RAG1 is defined as the RAG1 molecule that performs catalysis on the bound RSSs. The main chain or side chain of the residues in RNH that have potential interaction with DNA are shown as sticks (light blue). The nucleotide to be nicked is highlighted in cyan. c, Active site in the RNH region (shown in yellow) for nicked RSS (shown as sticks). The heptamers and coding flanks are shown in salmon and light cyan, respectively. The nucleotides to be nicked are highlighted in cyan. The metal ions (A and B) in the active centers are shown as orange spheres and residues that coordinate the metal ions are highlighted in light blue. Dashed black lines indicate potential coordination between metal ions and ligands. d, Superposition of the RNH domains that are bound to melted and nicked RSSs showing that the RNH active sites remain the same whether they are poised for nicking or hairpin formation. Bound RSSs are omitted. The zoom-in view displays the active site residues and metal ions.

Supplementary Figure 7 Detailed interactions of the ID with unmelted and melted DNAs in the coding flank–RSS junction.

a, Superposition of the 1st RAG1 that binds unmelted (colored as in Fig. 1a), melted (green) and nicked (cyan) RSSs respectively, and of Apo-RAG (gray). The 1st RAG1 is defined as the RAG1 molecule that performs catalysis on the bound RSSs. The α15 helices are indicated by black arrows. b,c, Detailed interactions of the ID (magenta) of the 1st RAG with unmelted RSS (pale cyan) (b) and melted RSS (pale cyan) (c) with heptamer highlighted in salmon. The main chain or side chain of the residues in ID that have potential interaction with DNA are shown as sticks (light blue). The nucleotide that is potentially recognized specifically by RAG is highlighted in red.

Supplementary Figure 8 Interactions of the β4–β5 loop with different DNAs.

a,b, Detailed interactions of the β4-β5 loop (lemon) from the 2nd RAG1 with melted DNA (a) and nicked DNA (b), respectively. The 2nd RAG1 is defined as the RAG1 molecule that only mediates the binding of the RSS but not catalysis. Heptamers are highlighted in salmon. The main chains or side chains of residues in the β4-β5 loop that have potential interactions with the DNA are shown as sticks (light blue). The nucleotides to be nicked or cleaved are highlighted in cyan.

Supplementary Figure 9 Structural and mechanistic implications for DNA transposases.

a, Superposition of the apo-Hermes dimer (2BW3) and the Hermes dimer bound to inverted repeats (light blue) (4D1Q) by aligning one Hermes molecule (left). Only the RNH and ID domains in Hermes are shown. The RNH domains in apo-Hermes and Hermes bound to DNA are shown in pale green and yellow, respectively, and the ID domains in apo-Hermes and Hermes bound to DNA are shown in gray and pink, respectively. The difference at the second Hermes monomer between the apo form and the synaptic complex form is evident (red arrow). b, Superposition of the inactive Tn5 dimer (1B7E) and the Tn5 dimer bound to hairpin mimic DNA (light blue) (1MUH) by aligning one Tn5 molecule (left). Only the RNH and ID domains in Tn5 are shown. The RNH domains in apo-Tn5 and Tn5 bound to hairpin mimic DNA are shown in pale green and yellow, respectively, and the ID domains in apo-Tn5 and Tn5 bound to hairpin mimic DNA are shown in gray and pink, respectively. The difference at the second Tn5 monomer between the apo form and the synaptic complex form is evident (red arrow). c, Examples of the signal or inverted repeat sequences in RAG and other DNA transposases and integrases. The flanking sequences are shown in lower case and the signal or IR sequences are shown in upper case. The conserved di-nucleotides CA and GT around the cleavage sites are highlighted in red. MuA: transposase of the bacteriophage Mu; Mos1: transposase in the Mariner family; PFV: prototype foamy virus integrase.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9, Supplementary Note and Supplementary Table 1

Reporting Summary

Supplementary Video 1

A rotational view of RAG in complex with one unmelted RSS and one melted RSS. The RAG structure in complex with melted 12-RSS and unmelted 23-RSS (PDB ID 6DBV) is shown rotating around a vertical axis that is close to the 2-fold axis of the core region of the RAG1–RAG2 complex.

Supplementary Video 2

A composite morph from apo-RAG to RAG in complex with doubly unmelted RSS, to RAG in complex with one melted RSS and one unmelted RSS, and to RAG in complex with doubly melted RSS. The zebrafish apo-RAG structure was modeled from PDB ID 4WWX, and PDB IDs for zebrafish RAG structures with doubly unmelted RSS, one melted RSS and one unmelted RSS, and doubly melted RSS are 6DBT, 6DBV, and 6DBL respectively.

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Ru, H., Mi, W., Zhang, P. et al. DNA melting initiates the RAG catalytic pathway. Nat Struct Mol Biol 25, 732–742 (2018). https://doi.org/10.1038/s41594-018-0098-5

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