A single enzyme active site that catalyzes multiple reactions is a well-established biochemical theme, but how one nuclease site cleaves both DNA strands of a double helix has not been well understood. In analyzing site-specific DNA cleavage by the mammalian RAG1–RAG2 recombinase, which initiates V(D)J recombination, we find that the active site is reconfigured for the two consecutive reactions and the DNA double helix adopts drastically different structures. For initial nicking of the DNA, a locally unwound and unpaired DNA duplex forms a zipper via alternating interstrand base stacking, rather than melting as generally thought. The second strand cleavage and formation of a hairpin–DNA product requires a global scissor-like movement of protein and DNA, delivering the scissile phosphate into the rearranged active site.
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The accession numbers for the cryo-EM structures and associated density maps of the mouse PRC and NFC complexes reported in this paper have been deposited to the PDB and EMDB under accession codes PDB 6OEM to 6OER and 6V0V and EMD-20030 to EMD-20035, EMD-20038, EMD-20039 and EMD-21003, as specified in Table 1.
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This research was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (M.G., DK036167; W.Y., DK036147 and DK036144; Z.H.Z., GM071940). The authors acknowledge the use of instruments at the Electron Imaging Center for NanoMachines supported by the NIH (1S10RR23057, 1S10OD018111 and U24GM116792), NSF (DBI-1338135 and DMR-1548924) and CNSI at UCLA.
The authors declare no competing interests.
Peer review information Anke Sparmann was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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a, b, Cleavage efficiencies (nicking and hairpinning) of the three DNA variants by WT mRAG at 22 and 37 °C (mean and s.d., n= 3 independent samples). c, Percentage of NFC and PRC (NFC/PRC) in cryo-EM 3D classification from samples made of DNA0, DNA1 or DNA2 substrate with E962Q mutant mRAG at 22 and 37 °C. Asterisk (*) indicates that the dataset was collected on a Tecnai F20 electron microscope instead of Titan Krios.
a, Flow chart for cryo-EM data processing of mRAG complexed with DNA1. The maps with red bold letter are used for final model building. b, A surface presentation of the 3.7 Å NFC (DNA1) map (C1 symmetry). Colors are according to the local resolution estimated by ResMap, and the color scale bar is shown on its right. c, Angular distributions of all particles used for the final three-dimensional reconstruction shown in b. d, The FSC curves of the NFC (DNA1) map (C1). The “gold standard” FSC between two independent halves of the map (black line) indicates a resolution of 3.7 Å, and the blue line is the FSC between the final refined model and the final map. e to i, Representative regions of the C1 map (transparent grey surface). The maps are shown with the final structural models (cartoon or stick) superimposed.
a, Flow chart for cryo-EM data processing of mRAG complexed with DNA2. The maps with red bold letter are used for final model building. b, A surface presentation of the 3.3 Å NFC (DNA2) map (C1 symmetry). Colors are according to the local resolution estimated by ResMap, and the color scale bar is shown on its right. c, Angular distributions of all particles used for the final three-dimensional reconstruction shown in b. d, The FSC curves of NFC (DNA2) map (C1). The “gold standard” FSC between two independent halves of the map (black line) indicates a resolution of 3.3 Å, and the blue line is the FSC between the final refined model and the final map. e to i, Representative regions of the C1 map (transparent grey surface). The maps are shown with the final structural models (cartoon or stick) superimposed.
a, Cryo-EM structures of NFC with DNA1 (green) and DNA2 (blue) are superimposable. b, Comparison of mouse PRC and NFC structures. Superposition of cryo-EM PRC (red) and NFC (DNA1) (green) structures reveals limited NBD and nonamer movement, which is marked with blue dashed circle (right panel). c, Superposition of crystal (grey) and cryo-EM (red) PRC structures reveals the different NBD and nonamer region (circled in red dashes) due to crystal-lattice contacts. d, e, The zoom-in views of the active center and DNA distortions in the superimposed structures shown in a-b. The catalytic DDE motif and two metal ions (a and b) are labeled; the heptamer of RSS DNA is shown in detailed cartoon presentation; the scissile phosphate of top strand is marked by a large ball. In panel d, bases forming the DNA zipper are labeled. f, A zoom-in view of boxed area in panel b. RAG2 interacts with the minor groove in PRC or the major groove in NFC.
a, Flow chart for cryo-EM data processing of WT mRAG complexed with DNA0. Data processing was done using RELION. The 3.6Å map labeled in red was used for final model building. b, A surface presentation of the 3.6 Å map of WT NFC (DNA0). Colors are according to the local resolution estimated by ResMap, and the color scale bar is shown on its right. c, Angular distributions of all particles used for the final three-dimensional reconstruction. d, The FSC curves of WT NFC (DNA0) map. The “gold standard” FSC between two independent halves of the map indicates an overall resolution of 3.6 Å. e–h, Representative regions of the map (transparent grey surface). The refined zipper DNA fits the map better (e) than the melted DNA (PDB: 6DBR) (f).
a, b, Unbiased simulations were run starting from the base-flipped out structure (PDB 6DBR) (a), and the zipper structure (b) using the Amber 14 force field. Plotted in each case are the all-atom RMSD to the base-flipped out structure (black) and the zippered structure (red). The structures at the start and end of each run are shown above the RMSD plots. c, d, The analogous results are given for simulations with the CHARMM 36 force field. e, Biased simulations were run from a canonical B-DNA form, in which the terminal residues were driven to mimic the stretched and untwisted DNA observed in the mouse and zebrafish NFC structures. The RMSD and initial and final structures are shown as before.
a, Flow chart for the cryo-EM data processing of mRAG complexed with DNA0. The maps with red bold letter are used for final model building. b, A surface presentation of the 3.6 Å NFC map (C1 symmetry). Colors are according to the local resolution estimated by ResMap, and the color scale bar is shown on its right. c, Angular distributions of all particles used for the final three-dimensional reconstruction. d, The FSC curves of PRC (DNA0) map (C1). The “gold standard” FSC between two independent halves of the map (black line) indicates a resolution of 3.6 Å, and the blue line is the FSC between the final refined model and the final map. e to i, Representative regions of the C1 map (transparent grey surface). The maps are shown with the final structural models (cartoon or stick) superimposed. j, The maps of 23RSS in PRC (DNA0), NFC (DNA1) and NFC (DNA2).
a, Repositioning of αX in the RNH domain during PRC to NFC transition (both are cryo-EM structures). E962 is far from the active site in PRC (light blue) but is positioned for catalysis in NFC (green). b, Anomalous X-ray scattering of the PRC crystals confirms that one Mn2+ and one Zn2+ are bound to each RAG1 subunit. The anomalous map is contoured at 3σ in red. The blue 2Fo-Fc map (contoured at 1σ) highlights R848, which is buried in the minor groove. c, The reconfigured E962 in HFC (pink) after the first DNA cleavage by nicking (green).
Supplementary Fig. 1 and Supplementary Table 1.
DNA zipper formation. The two base pairs (AC/(TG)) undergoing zipper formation are shown as sticks, and the rest of the DNA is simplified as a tube and ladder cartoon. The active site DDE are shown in red sticks, and divalent cations are represented as green spheres. The second Mg2+ ion appears only at the end because it binds in a fully formed active site with the substrate in place, but not in PRC. The red sphere on the DNA strand marks the scissile phosphate for nicking, and the DNA becomes nicked when two Mg2+ ions are properly bound.
Morphing of structural transition from PRC to NFC. The mRAG protein is shown as a semitransparent molecular surface. ZnH2 domains (green and light blue cartoon) of RAG1 move significantly and two L12 loops (green and light blue coils) become extended. The second metal ion (green sphere) is captured in the active site (marked by D600, D708 and E962 in red sticks) only when E962 and the scissile phosphate are in the active configuration. The 12/23RSS DNAs are shown as yellow and orange tube-and-ladders.
Morphing of the structural transition from NFC to HFC. One RAG1–RAG2 heterodimer (bound to 12RSS, yellow) is superimposed between the two structures and shown as a semitransparent molecular surface. The second RAG1–RAG2 heterodimer (bound to 23RSS, orange), shown as a colored cartoon, moves towards RAG-12RSS, delivering the bottom strand on each RSS DNA into the active site. Two L12 loops (green and light blue coil) of RAG1 subunits move away from the RAG1 interface. The configuration of the active center (red stick-and-balls for catalytic residues), except for E962, remains unchanged, and the closing motion positions the scissile phosphates on the bottom strands into the active centers. The green spheres represent Mg2+ ions. The second Mg2+ ion appears only when the active site is fully formed, but not when DNA undergoes conformational changes. When both Mg2+ ions are properly bound in the active site, the DNA cleavage/transesterification reaction takes place as shown.
The reaction cycle of DNA nicking and hairpin formation catalyzed by RAG recombinase. The scissile phosphates for nicking and hairpinning of DNA1 are highlighted by red and pink spheres. The active site DDE are shown as red sticks, and two metal ions in the NFC and HFC states are shown as green spheres. When the catalytic carboxylates DDE are not fully aligned or the scissile phosphate is not captured in the active site, such as during conformational changes of RAG and DNA, only one metal ion may be retained.
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Chen, X., Cui, Y., Best, R.B. et al. Cutting antiparallel DNA strands in a single active site. Nat Struct Mol Biol 27, 119–126 (2020). https://doi.org/10.1038/s41594-019-0363-2
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