How mouse RAG recombinase avoids DNA transposition

Abstract

The RAG1-RAG2 recombinase (RAG) cleaves DNA to initiate V(D)J recombination, but RAG also belongs to the RNH-type transposase family. To learn how RAG-catalyzed transposition is inhibited in developing lymphocytes, we determined the structure of a DNA-strand transfer complex of mouse RAG at 3.1-Å resolution. The target DNA is a T form (T for transpositional target), which contains two >80° kinks towards the minor groove, only 3 bp apart. RAG2, a late evolutionary addition in V(D)J recombination, appears to enforce the sharp kinks and additional inter-segment twisting in target DNA and thus attenuates unwanted transposition. In contrast to strand transfer complexes of genuine transposases, where severe kinks occur at the integration sites of target DNA and thus prevent the reverse reaction, the sharp kink with RAG is 1 bp away from the integration site. As a result, RAG efficiently catalyzes the disintegration reaction that restores the RSS (donor) and target DNA.

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Fig. 1: Similarity of hairpin formation and disintegration catalyzed by RAG.
Fig. 2: mRAG-DNA interactions in the STC.
Fig. 3: The T-form DNA in STC.
Fig. 4: Comparison of three different U-shaped DNAs.
Fig. 5: Distorted target DNA in transposition.
Fig. 6: RAG2 enforces the target DNA distortion.

Data availability

The cryo-EM structures of STC are available from the Protein Data Bank under accession codes 6OES and 6OET, and the associated density maps are available under codes EMD-20036 and EMD-20037 from the Electron Microscopy Data Bank (Table 1).

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Acknowledgements

W.Y. is grateful to W. Olson and S. Li for analyzing the T-form DNA structure. 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). We acknowledge the use of instruments at the Electron Imaging Center for NanoMachines supported by NIH (1S10RR23057, 1S10OD018111 and U24GM116792), NSF (DBI-1338135 and DMR-1548924) and CNSI at UCLA.

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Contributions

X.C. carried out all experiments and structure determination. Y.C. collected cryo-EM micrographs on the Krios microscope at UCLA and helped with structure determination and refinement. H.W. helped with cryo-EM data collection on the TF20 and Krios systems at NIH. Z.H.Z., W.Y. and M.G. supervised the research project. X.C., M.G. and W.Y. prepared the manuscript.

Corresponding authors

Correspondence to Martin Gellert or Wei Yang.

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Peer review information Beth Moorefield 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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Extended data

Extended Data Fig. 1 Two types of DNA cleavage mechanism used by RNase H-like transposases.

a, RAG and members of eukaryotic hAT transposase family, e.g. Hermes, cleave the top strand and generate a 5′ phosphate on the transposon end (terminal inverted repeat, TIR), or recombination signal sequence (RSS for RAG) first. Cleavage of the bottom strand occurs by hairpin formation on DNA flanking the TIR or RSS. The filled and open red circles indicate the scissile phosphates of the top and bottom strand, respectively. b, All bacterial and many eukaryotic transposases including retroviral integrases cleave the bottom strand first and generate a 3′-OH on the transposon end for transposition. The pink arrow before the hairpin formation step and the dashed grey box indicate that only a subset of transposases in this class undergo hairpin formation. The site of first nick is marked by a red scissor in a and b, and the transposition competent complexes are shaded. c, Target capture and strand transfer reaction. The target site in T-DNA, which is duplicated after transposition, is shown as a base pair ladder, and nucleophilic attack is indicated by red arrows.

Extended Data Fig. 2 Structure determination of RAG STC by cryo-EM.

a, Flow chart for the cryo-EM data processing. The maps with red bold letters are used for final model building of an intact STC and focused refinement without NBD and nonamer regions (STC∆NBD). b,c, A representative cryo-EM micrograph (b) and 2D classes of different views (c). d, A surface presentation of the 3.06 Å STC∆NBD map (C1 symmetry). Colors are according to the local resolution estimated by ResMap, and the color scale bar is shown on its right. e, Angular distributions of all particles used for the final three-dimensional reconstruction shown in b. f, The FSC curves of STC map (C1). The “gold standard” FSC between two independent halves of the map (black line) indicates a resolution of 3.06 Å, and the blue line is the FSC between the final refined model and the final map. g, Directional FSC plots54 of the cryo-EM reconstruction of STC∆NBD. h-k, Representative regions of the 3.06 Å STC∆NBD map (transparent grey surface). The maps of αX helix (h) heptamer plus one Ca2+ (i) L12 in RNH domain (j) and target DNA (k) are shown with the final structural models (cartoon or stick) superimposed.

Extended Data Fig. 3 Disintegration reaction is inhibited in RNH-type transposases.

a,b. Similarity between the hairpin formation in HFC (a) and disintegration in STC (b) catalyzed by RAG. The DNAs are colored in yellow (RSS), orange (the coding flank in HFC), and pink (the flank) and purple (the 5 bp target) of T-form DNA in STC. The RAG active site is marked by two divalent cations, shown as green spheres. The nucleophilic reaction is indicated by a red arrow. ce, The reaction center for disintegration in RAG, PFV (PDB: 4BAC) and MuA (PDB: 4FCY). In the RAG STC (c) the 3′-OH nucleophile (in a dashed circle) is aligned for disintegration, but in the PFV STC (d) the entire nucleotide at the 3′-end is misaligned relative to the scissile phosphate. The direction of nucleophilic attack is marked by the dotted red arrow. In the MuA STC (e) the 75° kink at the integration site renders the 3´ end 15.1 Å away from the scissile phosphate.

Extended Data Fig. 4 Mild DNA distortion in complex with Cas1-Cas2.

The spacer is equivalent to the transposon DNA in transposition (TIR or RSS) and is colored in yellow. The repeat is equivalent to the target DNA in transposition and colored green. Because the target site is more than 20 bp, the repeat DNA is bent gently in the middle and far from the DNA integration sites.

Supplementary information

41594_2019_366_MOESM3_ESM.mov

The animation shows a 90° rotation view of the T-form target DNA in the RAG STC complex. To deform the B-DNA to T-DNA requires kinking the B-DNA twice 3 bp apart by 85° towards the minor groove and then further twisting the flank DNA segments relative to the central three distorted base pairs. The two steps of DNA distortion are shown in three orthogonal views. The gray balls indicate the transposon DNA insertion (or integration) sites.

Supplementary Information

Supplementary Fig. 1 and Supplementary Table 1.

Reporting Summary

Supplementary Video 1

The animation shows a 90° rotation view of the T-form target DNA in the RAG STC complex. To deform the B-DNA to T-DNA requires kinking the B-DNA twice 3 bp apart by 85° towards the minor groove and then further twisting the flank DNA segments relative to the central three distorted base pairs. The two steps of DNA distortion are shown in three orthogonal views. The gray balls indicate the transposon DNA insertion (or integration) sites.

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Chen, X., Cui, Y., Wang, H. et al. How mouse RAG recombinase avoids DNA transposition. Nat Struct Mol Biol 27, 127–133 (2020). https://doi.org/10.1038/s41594-019-0366-z

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