How RAG1/2 evolved from ancestral transposases to initiate V(D)J recombination without transposition

How RAG1/2 evolved from ancestral transposases to initiate V(D)J recombination without transposition | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article How RAG1/2 evolved from ancestral transposases to initiate V(D)J recombination without transposition Xuemin Chen, Liangrui Yao, Shanshan Ma, Xingyun Yuan, Yang Yang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5443361/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Jul, 2025 Read the published version in Proceedings of the National Academy of Sciences → Version 1 posted You are reading this latest preprint version Abstract The RAG1/2 recombinase, which initiates V(D)J recombination in jawed vertebrates, evolved from RNaseH-like transposases such as Transib and ProtoRAG 1 . However, its post-cleavage transposase activity is strictly suppressed. Previous structural studies have focused only on the conserved core domains of RAG1/2, leaving the regulatory mechanisms of the non-core regions unclear. To investigate how RAG1/2 suppresses transposition and regulates DNA cleavage, we determined cryo-EM structures of nearly full-length RAG1/2 complexed with cleaved Recombination Signal Sequences (RSS) in a Signal-End Complex (SEC), at resolutions up to 2.95 Å. Two key structures, SEC-0 and SEC-PHD, reveal distinct regulatory roles of RAG2, which is absent in Transib transposase. SEC-0 displays a closed conformation, revealing that the core RAG2 facilitates sequential DNA cleavage by stabilizing the RSS-cleaved states in a "spring-loaded" mechanism. SEC-PHD reveals how RAG2’s non-core PHD and Acidic Hinge (AH) domains, which are absent in ProtoRAG, inhibit target DNA binding in transposition. Histone H3K4me3, which recruits RAG1/2 to RSS sites, does not influence RAG1/2 binding to V, D or J gene segments bordered by RSS 2 . In contrast, the suppressed transposition can be activated by H3K4me3 peptides that dislodge the inhibitory PHD domain 3,4 . To achieve this de-repression in vivo, however, would require an unlikely close placement of two nucleosomes flanking a target DNA bent by nearly 180°. Our structural and biochemical results elucidate how RAG1 has acquired RAG2 and utilizes its core and non-core domains to enhance V(D)J recombination and suppress transposition. Biological sciences/Biochemistry/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Molecular biology/DNA recombination Biological sciences/Biochemistry/Enzymes/Recombinases RAG transposition target DNA PHD H3K4me3 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction V(D)J recombination is the essential process generating the adaptive immune system with both diversity and specificity to neutralize a great variety of infectious agents 5 . RAG1/2 (recombination activating genes 1 and 2) protein, a heterotetramer of two RAG1 and two RAG2 subunits, cleaves at the boundaries of RSSs (recombination signal sequences) and flanking V, D and J gene segments of antigen receptors by first nicking and then hairpinning, and the resulting blunt-end signal ends and hairpin-end gene segments (also known as coding flanks) are separately re-joined by Non-Homologous End Joining (NHEJ) (Fig. 1a) 1,5,6 . The core and non-core domains of RAG are respectively responsible for the DNA cleavage and the regulation of V(D)J recombination (Fig. 1b). RAG1 has an RNase H-like (RNH) catalytic core domain (aa 384-1008) 6,7 . Like all RNH-containing transposases, from bacterial Tn5, MuA, Drosophila P element, to eukaryotic Hermes and retroviral integrases 8-11 , RAG1/2 catalytic core not only cleaves at RSS, but also can insert the two cleaved RSS ends as Terminal Inverted Repeats of transposon ends (TIR) into a new 5-bp GC-rich target site in vitro or ex vivo 12,13 . However, the deleterious transposition by RAG1/2 is suppressed in vivo 14,15 . RAG2 is absent in transposases including Transib. Beyond its core domain (aa 1-387), the regulatory PHD (aa 410-485) and Acidic Hinge (AH, aa 388-409), which are only present in jawed vertebrates and absent in RAG2L of protoRAG, have been reported to suppress transposition 16,17 . The RAG2 PHD domain is also necessary for recruiting RAG1/2 to V, D and J gene segments on open chromatin by its binding to a histone H3K4me3 tail 18 . Interestingly, the PHD domain also moderately autoinhibits RSS DNA cleavage, and a 21-aa H3K4me3 peptide can release this inhibition 3,19 . The mouse apo-RAG1/2 (mRAG) was the first core RAG structure determined 20 . Subsequently, structures of RSS DNA binding, cleavage, and even post-cleavage transposition of RSS into a target DNA (tDNA), have been reported for mRAG, zRAG (zebrafish), Transib, and ProtoRAG 7,17,21-27 . From DNA binding (pre-reaction complex or PRC), nick-forming (NFC) to hairpin-forming complex (HFC), RAG and related recombinases undergo an open-to-close conformational change between its two halves in the DNA cleavage process (Fig. 1a). The same closed conformation is maintained by mRAG in the strand transfer complex (STC), in which the tDNA is kinked twice by 85° (170° overall) 3 bp apart in the 5 bp 5ʹ-CGCCG-3ʹ target site and a further twist out of plane 26 . The severe distortion of tDNA likely hinders RAG-mediated transposition and promotes robust disintegration to revert STC to SEC and tDNA 26,27 . The structure of the core RAG complexed with cleaved RSS signal ends, without coding flanks (SEC), has eluded characterization, and it is unclear if mRAG SEC remains closed or becomes open like Transib bound only to the cleaved TIRs 24 . When the PHD domain of RAG2 was included in structural studies of V(D)J recombination, a histone H3K4me3 peptide was always added to activate DNA cleavage 21,23,25-27 . As a result, PHD and the other non-core domains of RAG are disordered in all RAG structures reported to date. In the SEC mimic of zRAG in the presence of PHD and H3K4me3, the pre-cleaved RSS DNA occupies the coding flank-binding sites and resembles the HFC structure 21 . How RAG2’s PHD inhibits RSS DNA cleavage and transposition remains unknown. Here, we assembled mRAG SEC with the near full-length protein and the pre-cleaved RSS DNAs in the absence of any H3K4me3 peptide, determined the cryo-EM structures at up to 2.95 Å resolutions, and identified a true SEC (SEC-0) and SEC bound by PHD (SEC-PHD) (Fig. 1 and Extended Data Fig. 1). These previously unknown structures illuminate how RAG2 supports the “spring-loaded” RSS-DNA cleavage and suppresses unwanted transposition. Cryo-EM analysis of mRAG SEC The mRAG SEC was assembled and purified using size-exclusion chromatography (see Methods). Although the elution profile revealed a single peak. (Extended Data Fig. 2a) and SEC particles on cryo-EM grids appeared homogeneous, the volume surrounding the flank-DNA binding sites (abbreviated as flank binding site below) on 2D averages and initial 3D reconstructions was quite varied, indicating structural heterogeneity 21 (Extended Data Fig. 1). After local 3D classification of the flank binding sites, four major structural species were identified and refined. They are SEC-0, with completely empty flank binding sites (2.95 Å, C2 symmetry), SEC-1DNA, with one blunt-end RSS DNA in one of two flank binding sites (~3.4 Å), SEC-2DNA with two blunt-end DNAs in both sites (3.0 Å, C2 symmetry), and lastly SEC-PHD with a single PHD domain and AH occupying both flank binding sites (3.25 Å, C1 symmetry). The RAG structures in SEC-1DNA and SEC-2DNA are indistinguishable from mRAG HFC and zRAG bound to RSS DNAs 21,22 . For the SEC-PHD structure, the crystal structure of PHD (PDB: 2V83) was readily docked into the large volume (~4 Å resolution) occupying the flank binding site (Fig. 1d), while AH (aa 387-409) was docked as an extended peptide into the remaining volume, but not modeled with individual residues due to the poor resolution of 6 Å or worse (Fig. 1d). Structure of SEC-0 and implications for recombination and transposition SEC-0 as well as the other three SEC structures adopts the same closed conformation as HFC and STC, and the RAG1/2 protein chains are well superimposed among them (C α atoms of RAG1 aa 461-1008 and RAG2 aa 1-350, RMSDs < 0.55 Å). The closed conformation of SEC-0 without mediation of the flanking DNAs in HFC or STC is maintained by the RAG1 and RAG2 interactions across the two RAG1/2 heterodimers, which we term trans interactions. RAG1’s helix O and loop L QR interact with RAG2’s loop L F2F3 and helix α E4F1 in trans via extensive charge-charge interactions. For example, the K827, R828, K835 and K839 of RAG1 form salt bridges with E341, D334, and D310, and H313 of RAG2 (Fig. 2a and 2b). Even though ProtoRAG2L contains a core region similar to mRAG2, L F2F3 in RAG2L is much shorter than in mRAG2, and charged residues on L F2F3 and α E4F1 are not conserved. It can be expected that the trans RAG1/2L interactions are absent in ProtoRAG. RNH-like transposases, most of which are devoid of RAG2-equivalent subunit, can bind, distort and cleave substrate dsDNA independent of a high-energy co-factor. Instead, by a hypothesized “spring-loaded” mechanism, they may use the initial protein-DNA binding energy to support the subsequent conformational changes and DNA transactions 28 When the apo mRAG1/2 binds to substrate RSS DNAs to form the PRC, RAG1/2 becomes very open, as if a spring is loaded, and the RAG1-RAG2 trans interaction is absent (Supplementary video 1). PRC transforms to NFC by DNA unwinding and protein domain (ZnH2) closing to nick the first DNA strand, then to the most closed HFC to cleave the second DNA strand by forming a hairpin. The SEC-0 with flanking sites empty retains the closed conformation of HFC, indicating that this closed form is the preferred and energy minimum state. Most transposases including Transib accomplish the two-step cleavage reaction without the help of RAG2, but the trans RAG1-RAG2 interactions and the resulting closed conformation of SEC-0 demonstrate how RAG2 supports and guides RAG1 to accomplish the “spring-loaded” mechanism in sequential DNA cleavage. While the catalytic residues reside entirely in RAG1, RAG2 becomes an essential accessary subunit to RAG1 by stabilizing RAG1 protein and enabling its “spring-loaded” mechanism of DNA cleavage 20,29,30 . The closed SEC renders transposition by RAG1/2 unlikely because for it to capture a target DNA, the tDNA has to either be already deformed by two 85° kinks 3 bp apart (Extended Data Fig. 4a) or wait for SEC to transiently open with all trans interactions broken. Neither scenario is of high probability. Unlike RAG1/2 recombinase, Transib lacks the RAG2 subunits, and its Transposon End Complex (TEC, equivalent to SEC) is much more open than SEC 24 (Fig. 2c-f and Extended Data Fig. 3). To be captured by Transib, a tDNA would need to be bent by 120° instead of 170° as by mRAG. When Transib carries out transposition, from TEC to STC, it undergoes a 30° closing motion because both Transib subunits bind each flank DNA, and the cleaved tDNA is bent 150°, 30° more than the intact tDNA (Fig. 2c). For RAG1/2, SEC and STC are superimposable (Fig. 2d), and a tDNA needs to be bent 170° before transposition can take place. A novel PHD-AH binding site revealed by SEC-PHD In the SEC-PHD structure, one PHD domain and the adjacent AH of RAG2 occupy the space vacated by the flank DNAs (Fig. 3a and 3b). The PHD occupies one flank binding site (called “RAG1/2 (a)”), and the AH fills the other (called “RAG1/2(b)”). These interactions are mediated by positively charged surfaces of the core RAG1/2 heterodimers and the negatively charged PHD and AH (Fig. 3c, 3d and Extended Data Fig. 2b, 2c). Loop T418-V425 of PHD interacts with the ZnH2 domain of RAG1(a). The side chains of T418 and D424 form hydrogen bonds and a salt bridge with R826 and R734, respectively (Fig. 3c). All backbone carbonyl oxygens except for D424 form polar interactions with R826, K806, R927, K931 and R734. Additionally, S435 of PHD hydrogen bonds with RAG2(a) residues H10 and N11, while N428 interacts with RAG1(a) R848 and M849. V431 interacts with RAG1(a) loop 720-722, and F433 forms a π-cation interaction with RAG2(a) R39 (Fig. 3c). Although the low-resolution map doesn’t permit a precise model of AH, aa 394-410 can be confidently located inside the positively charged tunnel lined by K823, R826, K806, R927, K931 and R734 on the RAG1/2(b), suggesting that AH imposes additional hindrance for tDNA binding (Fig. 3d). Because the PHD domain is wider than a DNA duplex, the flank binding site is remodeled to accommodate it by opening Helix O and loop L NO (aa 816-832) in the ZnH2 domain of RAG1(a) (Fig. 3e, Extended Data Fig. 4). In RAG1(a), the peptide between K817 and H818 is flipped, thus changing the conformation of loop L NO and pulling it outward, which is accompanied by a 9.7° and 1.6 Å bending of a part of helix O (aa 822-832). In contrast, binding of the AH to the core RAG1 doesn’t require any conformational changes, and RAG1(b) is superimposable with SEC-0. Because one of the two PHD domains and its adjacent AH of SEC-0 occupy both flank-DNA binding sites and block tDNA from entering SEC, to eliminate the asymmetric inhibition would require eliminating binding of both PHD domains and linked AHs. RAG2’s PHD has been shown to bind an H3K4me3 peptide and release the autoinhibition of RSS DNA cleavage by PHD domains 4-6 . It has been hypothesized that binding of H3K4me3 changes PHD structure and prevents PHD from associating with the core RAG1/2 4 . To our surprise, PHD domains in SEC-PHD and in the PHD-H3K4me3 complex (PDB: 2V83) are superimposable with an RMSD of 0.687 Å over 58 pairs of C α atoms, and H3K4me3 binding appears to stabilize the otherwise flexible loop aa 471-475 in PHD domain (Fig. 4a, 4b). The buried surface of PHD in the PHD-H3K4me3 complex (735 Å 2 ) is smaller than that in SEC-PHD (1,230 Å 2 ), and thus H3K4me3 cannot effectively compete with SEC for PHD binding. However, we note that the H3K4me3 peptide bound to PHD in the flank-binding site would clash with the core RAG2, starting from Ala7 of histone H3 onward 3,4 . This suggests a H3K4me3 peptide 6-residue in length would fail to release the inhibition of RSS DNA cleavage and transposition, and only longer peptides, as in nucleosomes, may dislodge and prevent the PHD from occupying the flank binding site. Interestingly, in the apo mRAG structure, the two halves of RAG1/2 core are oriented such that even a single PHD domain cannot fit (Extended Data Fig. 4e). This suggests that PHD domains cannot inhibit initial binding of V, D and J segments to RAG1/2 for RSS cleavage. Inhibition of DNA cleavage and transposition by PHD To assess whether the PHD domain inhibits PRC formation (initial V, D and J substrate binding), DNA nicking, hairpinning, and transposing, and whether long (21 aa) or short (6 aa) H3K4me3 peptides can dislodge and prevent PHD from binding and inhibiting the catalytic activities of core RAG1/2, we performed DNA cleavage and transposition assays with near full-length RAG1/2, and with or without H3K4me3 peptides. Using pre-nicked or pre-cleaved RSSs to compare DNA hairpinning activity or transposition of SEC into supercoiled circular DNA target, respectively, we found that the short peptide had no effect in both reactions; but the long peptide increased the final hairpinning product by less than 2-fold and transposition product by 22-fold (double-ended joint) (Fig. 4c, 4d). First, these results confirm that our hypothesis based on the SEC-PHD structure, and the release of autoinhibition of PHD on the core RAG1/2, depends on the steric clashes between a long methylated H3 tail (such as on a nucleosome) and the RAG1/2 core region. Second, the more than 10-fold different activation levels of the long H3 peptide on hairpinning and transposition likely reflect different autoinhibition mechanisms. V, D and J segments are connected to RSS DNA in cis , but a tDNA is a separate piece of DNA from RSS and thus in trans . Moreover, V, D and J segments do not have to bend to bind to RAG1/2 and form the PRC, unlike a tDNA to SEC. Therefore, we used uncut RSS-bordered V and J-like segments to test initial DNA binding and nicking activity. As predicted, no differences were observed among the 3 tested groups, including with or without the 21-aa H3K4me3 (Fig. 4e and Extended Data Fig. 5). In fact, the little to no alteration of initial binding and nicking in the presence of PHD and lack of stimulation by H3K4me3 peptides have been reported previously 19,31 . In this test, we also confirm a less than 2-fold increase of hairpinning product in the presence of the long H3K4me peptide (Extended Data Fig. 5). Indeed, PHD cannot inhibit V, D or J segment binding and nicking of the first strand because binding of RSS DNA results in occupation of flank-binding sites by the coding flanks, and PHD has no chance to compete. A nicked DNA is less rigid than an intact DNA, and thus PHD free of H3K4me3 can interfere with the alignment of nicked DNA substrate for hairpin formation. But the cis nature of V(D)J recombination renders the inhibitory effect of PHD mild and therefore activation by H3K4me3 rather moderate compared with its effect on transposition. We deduce that acquisition of PHD serves two purposes. One is to recruit RAG1/2 to active and open chromatin domains to increase the substrate specificity of V(D)J recombination, and the second is to inhibit transposition. Conclusion With the SEC structures, we now have a complete presentation of RSS DNA cleavage and post-cleavage DNA transposition by RAG (Fig. 5 ). Acquisition of the core RAG2 during evolution as exemplified by ProtoRAG2L stabilizes the catalytic subunit. The later addition of the trans interaction as observed between core RAG1/2 subunits increases the “spring-loaded” DNA cleavage efficiency, and the resulting closed SEC structure presents a barrier to transposition by requiring a tDNA bent nearly 170° with further twist. The AH and PHD addition to the RAG2 core raises the anti-transposition barrier by precluding tDNA binding site in SEC, and the result is inhibition of double-end strand transfer by over 20-fold (Fig. 4 ). Remarkably, PHD and AH have no effect on the substrate binding and nicking steps in V(D)J recombination, and only mildly inhibit hairpin formation. RAG1/2 catalyzed processes also depend on transcription and recruitment to open chromatin via the PHD domains (Fig. 5 ). A pair of antigen receptor gene segments enabled by the associated nucleosomes can bind to a RAG1/2 recombinase without DNA deformation. But to dislodge two PHD domains alternately bound to an inhibited SEC, a tDNA needs to bring in two nucleosomes, one on each flanking side of the 170° bent target site, and steric clashes of such closely positioned nucleosomes and SEC may further inhibit transposition (Fig. 5 ). In short, the evolutionary process of RAG1/2 exemplifies how additional core and non-core domains of RAG2 eliminate unwanted transposition, while making the recombinase more specific and efficient. Methods Protein and DNA preparation Both mouse WT RAG1 (aa 265–1040) and T490A RAG2 (aa 1-520) were N-terminally tagged by His6-MBP fusion, co-expressed in HEK293T cells and purified as previously described 20 , 22 . An additional step of Mono Q anion exchange chromatography improved protein purity and removed DNA contamination. The buffer used in amylose affinity purification was 20 mM HEPES (pH 7.4), 500 mM KCl, 5% glycerol, 2 mM DTT, 0.5 mM EDTA. The salt in the protein eluate from the amylose column was diluted to 100 mM before loading onto a Mono Q column (GE Healthcare), which was pre-equilibrated with 20 mM HEPES (pH 7.4), 100 mM KCl, 5% glycerol, 2 mM DTT, 0.5 mM EDTA. mRAG was eluted by a linear gradient of 100–500 mM KCl. The purified mRAG was buffer-exchanged into storage buffer containing 20 mM HEPES (pH 7.4), 500 mM KCl, 20% glycerol, 0.1 mM EDTA, 2 mM DTT, concentrated to 6–8 mg/ml, and stored at -80°C. Human HMGB1 (amino acids 1–163) was prepared as reported previously 32 . 12- and 23-RSS DNAs used for structural analyses and biochemical assays (Supplementary table 1) were synthesized as ssDNA and purified using either PAGE or HPLC method (General Biol.). Gel purified oligonucleotides were loaded onto a Glen Gel-Pak column (Glen Research) and eluted in deionized H 2 O. DNA was annealed in an annealing buffer containing 20 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 50 mM NaCl in a Thermocycler. Cryo-EM sample preparation and data collection To prevent catalysis, we incubated WT mRAG, HMGB and DNAs in a Ca 2+ -containing buffer. Both RAG1 and RAG2 subunits contain a N terminal MBP-tag. MBP-mRAG protein, pre-cleaved 12- and 23-RSS signal end DNAs, and HMGB1 (aa 1-163) were mixed at 1:0.9–1.2:0.9–1.2:1.8–2.4 molar ratio in buffer containing 20 mM HEPES (pH 7.4), 100 mM KCl, 5 µM ZnCl 2 , 1 mM DTT, 5% glycerol and 5 mM CaCl 2 and incubated at 37°C for 10 min. The mixture was further purified at 4°C by size exclusion chromatography on a Superdex 200 Increase 10/300 GL column (GE Healthcare) in buffer containing 20 mM HEPES (pH 7.3), 100 mM KCl, 1% glycerol, 1 mM DTT, 5 mM CaCl 2 . The elution peak fractions were pooled and used for cryo-EM grid preparation. 3 µl of the purified SEC (0.3 mg/ml) was spotted on freshly glow-discharged (SuPro Coolglow) QUANTIFOIL R 1.2/1.3 (Cu, 300 mesh) grids at 22°C and blotted for 5 s. The frozen grids were stored in liquid nitrogen before use. For structure determination, the frozen grids were loaded into a Titan Krios electron microscope operated at 300 kV for automated image acquisition with SerialEM software, at the Multi-Institute Cryo-EM Facility (MICEF) of NIH. Movies were recorded on a Gatan K2 Summit direct electron detector using the super-resolution mode at 130K nominal magnification (calibrated pixel size of 1.06 Å at the sample level, corresponding to 0.53 Å in super-resolution mode) and defocus values ranging from − 0.8 to -3.0 µm. During data collection, the total dose was 70 e − /A 2 . The detailed collection statistics are shown in Table 1 . Structure analysis and model refinement Cryo-EM analysis was performed using CryoSPARC. All frames in each collected movie were aligned and summed using Patch Motion Correction, and CTF estimation were made using Patch CTF Estimation. Blob Picker and Template Picker were used for particle picking, and particles were extracted using a box size of 264 * 264 pixels. 2D classifications and 3D classifications were used to remove junk particles and select the most homogeneous particles for in-depth 3D structural analyses. The final 3D reconstruction for each class was done using Non-Uniform Refinement, and the resulting map was post-processed using DeepEMhancer 33 . All reported resolutions are based on the “gold standard” refinement procedure and the 0.143 Fourier Shell Correlation (FSC) criterion. Local resolution was estimated using Local Resolution Estimation. For model building, STC (PDB: 6OES) and PHD (PDB: 2V83) structures were used as initial models to fit into the maps using Chimera 34 , and the resulting models were manually adjusted and rebuilt according to the cryo-EM map in COOT 35 . Phenix real-space refinement was used to refine the models. The refinement statistics are shown in Table 1 . The detailed classifications and map qualities of different conformations of SECs are shown in the Supplemental Information (Extended Data Fig. 1 ). DNA cleavage and strand transfer assays The RSS DNA cleavage assays were performed in a reaction buffer containing 25 mM HEPES (pH 7.4), 100 mM KCl, 1 mM DTT, 0.1 mg/ml BSA, and 5 mM MgCl 2 . 200 nM each of FAM-labeled 12-RSS and 23-RSS DNAs (including coding flanks, either intact or pre-nicked, shown in Table S1) were incubated with 200 nM of heterotetrametric WT RAG, 400 nM HMGB1 and 1 µM H3K4me3 peptide (Genscript) at 37°C for 0 to 40 min. Reactions were stopped by adding an equal volume of formamide buffer (95% (v/v) formamide, 12 mM EDTA and 0.3% bromophenol blue) and heating at 95°C for 10 min. Cleavage products were separated by 19% TBE-urea PAGE, visualized and quantified using a Typhoon PhosphorImager (GE Healthcare). Plots of biochemical data show the mean ± SD from three independent experiments using Prism software. The strand transfer (transposition) assay was carried out as previously reported 13 , 26 . Briefly, signal-end complex (SEC) was first assembled by mixing WT, pre-cleaved 12- and 23- RSS signal ends without coding flank and HMGB1 at 1:1:1:2 molar ration in a pre-reaction buffer (25 mM HEPES (pH 7.4), 100 mM KCl, 1 mM DTT, and 0.2 mM CaCl 2 ) at 37°C for 10 min. The strand transfer rection was carried out by mixing 200 ng supercoiled pUC19 plasmid, 300 nM SEC with 20 µM H3K4me3 peptide in a reaction buffer (25 mM HEPES (pH 7.4), 100 mM KCl, 1 mM DTT, 0.1mg/ml BSA and 5 mM MgCl 2 ) and incubating at 37°C for 1 h. The reaction was stopped by adding 25 mM EDTA, and proteins were removed by incubation with 0.4 mg/ml Proteinase K for 30 min at 37°C. DNA products were resuspended in 15 ul loading buffer after ethanol precipitation and separated on a 1.5% agarose gel by electrophoresis. DNA bands were stained with ethidium bromide and quantified using a Typhoon Phosphorimager (GE Healthcare). Data from three independent experiments are averaged and shown with standard deviations using Prism software. Declarations Data and Software Availability The accession numbers for the cryo-EM structures and associated density maps of the mouse SEC-0, SEC-PHD, SEC-12DNA, SEC-23DNA, SEC-12/23DNA complexes reported in this paper have been deposited to the PDB and EMDB under accession PDB codes 9JPX, 9JPU, 9JTS, 9JTU and 9JQN, and EMD-61717, EMD-61715, EMD-61816 EMD-61817 and EMD-61730, as specified in Table 1. Competing interests The authors declare no competing interests. Author contributions X.C., M.G. and W.Y. conceived the project; X.C. and L.Y. carried out all experiments and structure determination; H.W. and L.L. helped with cryo-EM data collection; X.C., M.G. and W.Y. supervised the research project; X.C., M.G. and W.Y. prepared the manuscript; All authors participate in the discussions. Acknowledgements We thank the staff at the core facility of Institute of Health Sciences and Technology (Anhui University) for technical support. This research was supported by National Natural Science Foundation of China to X.C. (32371270) and Y.Y. (3220979), Anhui Province Outstanding Youth Fund to X.C. (2308085Y21), Natural Science Research Project of Anhui Educational Committee to X.C. (2022AH030010), and National Institute of Diabetes and Digestive and Kidney Diseases (USA) to M.G. (DK036167) and W.Y. (DK036147). References Liu C, Zhang Y, Liu CC, Schatz DG (2021) Structural insights into the evolution of the RAG recombinase. Nat Rev Immunol Jones JM, Simkus C (2009) The roles of the RAG1 and RAG2 non-core regions in V(D)J recombination and lymphocyte development. Arch Immunol Ther Exp (Warsz) 57:105–116 Matthews AG et al (2007) RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination. 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Nucleic Acids Res 22:1805–1809 Shimazaki N, Tsai AG, Lieber MR (2009) H3K4me3 stimulates the V(D)J RAG complex for both nicking and hairpinning in trans in addition to tethering in cis: implications for translocations. Mol Cell 34:535–544 Grundy GJ, Ramon-Maiques S, Dimitriadis EK, Kotova S, Biertumpfel C, Heymann JB, Steven AC, Gellert M, Yang W (2009) Initial stages of V(D)J recombination: the organization of RAG1/2 and RSS DNA in the postcleavage complex. Mol Cell 35 Sanchez-Garcia R et al (2021) DeepEMhancer: a deep learning solution for cryo-EM volume post-processing. Commun Biol 4:874 Pettersen EF et al (2004) UCSF Chimera–a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612 Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66:486–501 Tables Table 1. Statistics of cryo-EM data collection and structure refinement SEC-0 (EMDB: EMD-61717) (PDB: 9JPX) SEC-PHD (EMDB: EMD-61715) (PDB: 9JPU) SEC-1DNA (12RSS side) (EMDB: EMD-61816) (PDB: 9JTS) SEC-1DNA (23RSS side) (EMDB: EMD-61817) (PDB: 9JTU) SEC-2DNA (EMDB: EMD-61730) (PDB: 9JQN) Data collection and processing Magnification 130,000 130,000 130,000 130,000 130,000 Voltage (kV) 300 300 300 300 300 Electron exposure (e – /Å 2 ) 60 60 60 60 60 Defocus range (μm) -0.8 to -3.0 -0.8 to -3.0 -0.8 to -3.0 -0.8 to -3.0 -0.8 to -3.0 Pixel size (Å) 1.06 1.06 1.06 1.06 1.06 Symmetry imposed C2 C1 C1 C1 C2 Initial particle images (no.) 6,917,387 6,917,387 6,917,387 6,917,387 6,917,387 Final particle images (no.) 260,975 57,418 90,752 72,732 144,322 Map resolution (Å) (FSC threshold=0.143) 2.95 3.25 3.36 3.43 3.03 Refinement Initial model used (PDB code) 9JPU 6OES 9JPU 9JPU 9JPU Model resolution (Å) (FSC threshold=0.5) 3.2 3.6 3.7 3.7 3.3 Model composition Non-hydrogen atoms Protein residues Ligands (nucleotide) 15,399 1,784 58 15,963 1,853 58 18,710 1,928 164 18,710 1,928 164 16,488 1,784 112 B factors (Å 2 ) Protein Ligand (nucleotide) 64.77 77.33 77.87 96.33 73.18 152.32 72.61 148.34 56.11 93.12 R.m.s. deviations Bond lengths (Å) Bond angles (°) 0.539 0.006 0.471 0.002 0.579 0.003 0.575 0.003 0.587 0.003 Validation MolProbity score Clashscore Rotamers outliers (%) 1.94 6.12 2.29 1.78 6.39 1.84 1.81 10.24 0.18 1.85 10.41 0.12 1.78 9.07 0.26 Ramachandran plot Favored (%) Allowed (%) Disallowed (%) 95.32 4.68 0 96.51 3.43 0.05 95.92 4.08 0 95.45 4.55 0 95.71 4.12 0.17 Additional Declarations There is NO Competing Interest. Supplementary Files MovieS.mov Supplementary Video 1 RS.pdf Reporting Summary 9JPUSECPHD.zip 9JPU(SEC-PHD) 9JTUSEC1DNA23RSS.zip 9JTU(SEC-1DNA_23RSS) 9JPXSEC0.zip 9JPX(SEC-0) 9JTSSEC1DNA12RSS.zip 9JTS(SEC-1DNA_12RSS) 9JQNSEC2DNA.zip 9JQN(SEC-2DNA) ExtendedData.docx Cite Share Download PDF Status: Published Journal Publication published 28 Jul, 2025 Read the published version in Proceedings of the National Academy of Sciences → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5443361","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":404045146,"identity":"8225ed58-e64b-4c66-9eb4-c1016115a9f3","order_by":0,"name":"Xuemin Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYJACZih14MCHH6RpYUs8OLOHNC08xoc52IhQzj8jO/lzQcW9aP7ZPR8OM/AwyPOLHcCvReLM2W3SM84U5864c3bD4QILBsOZsxPwazFg793GzNuWkNtwI3fD4Rk8DAkGtwlpYebd/Jn3X0Lu/Bs5Dw7zsBGjhb13gzRvQ0Luhhs5DMRpAfuF51hC7sYbaQbAQJYg7Bf+GbmbP/PUJOTOu5H8+MOHHzby/NIEtGDYSpryUTAKRsEoGAXYAQDluEgAtIOrlgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-1818-9206","institution":"Anhui university","correspondingAuthor":true,"prefix":"","firstName":"Xuemin","middleName":"","lastName":"Chen","suffix":""},{"id":404045147,"identity":"bd114ef7-b50f-4f98-a6ce-0e05aaed9fcb","order_by":1,"name":"Liangrui Yao","email":"","orcid":"","institution":"Anhui university","correspondingAuthor":false,"prefix":"","firstName":"Liangrui","middleName":"","lastName":"Yao","suffix":""},{"id":404045148,"identity":"6461b2b7-bcdd-47ad-841b-567476ac7b5f","order_by":2,"name":"Shanshan Ma","email":"","orcid":"","institution":"Anhui university","correspondingAuthor":false,"prefix":"","firstName":"Shanshan","middleName":"","lastName":"Ma","suffix":""},{"id":404045149,"identity":"728fa6d2-751a-4fdf-bb2b-6541f3d577fb","order_by":3,"name":"Xingyun Yuan","email":"","orcid":"","institution":"Anhui university","correspondingAuthor":false,"prefix":"","firstName":"Xingyun","middleName":"","lastName":"Yuan","suffix":""},{"id":404045150,"identity":"fb33c720-b8ab-4195-b143-1a39014f1d14","order_by":4,"name":"Yang Yang","email":"","orcid":"","institution":"Anhui university","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Yang","suffix":""},{"id":404045151,"identity":"017bb011-99dc-41fd-960c-fe2bf773ec5a","order_by":5,"name":"Yuan Yuan","email":"","orcid":"","institution":"Anhui university","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Yuan","suffix":""},{"id":404045152,"identity":"6095c8c5-4b0d-437f-a297-0a085669d049","order_by":6,"name":"Yumei Liu","email":"","orcid":"","institution":"Anhui university","correspondingAuthor":false,"prefix":"","firstName":"Yumei","middleName":"","lastName":"Liu","suffix":""},{"id":404045153,"identity":"ce8048cc-2727-4bca-ae17-b2772e07ecf8","order_by":7,"name":"Lan Liu","email":"","orcid":"","institution":"National Institutes of Health","correspondingAuthor":false,"prefix":"","firstName":"Lan","middleName":"","lastName":"Liu","suffix":""},{"id":404045154,"identity":"acffa9ff-5a77-4433-8b98-c3072f63eae8","order_by":8,"name":"Huaibin Wang","email":"","orcid":"","institution":"National Institutes of Health","correspondingAuthor":false,"prefix":"","firstName":"Huaibin","middleName":"","lastName":"Wang","suffix":""},{"id":404045155,"identity":"28d872ea-1aeb-4a59-a767-a8816e060774","order_by":9,"name":"Wei Yang","email":"","orcid":"https://orcid.org/0000-0002-3591-2195","institution":"NIH/NIDDK","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Yang","suffix":""},{"id":404045156,"identity":"e779c6ff-44a5-4a3f-bba7-13f562d170f4","order_by":10,"name":"Martin Gellert","email":"","orcid":"https://orcid.org/0000-0002-0832-4929","institution":"National Institutes of Health","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"","lastName":"Gellert","suffix":""}],"badges":[],"createdAt":"2024-11-13 03:30:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5443361/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5443361/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1073/pnas.2512362122","type":"published","date":"2025-07-29T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":76112383,"identity":"b14e015c-2d71-41ac-a6ac-3ab02d41839c","added_by":"auto","created_at":"2025-02-12 12:10:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1140363,"visible":true,"origin":"","legend":"\u003cp\u003eV(D)J recombination and cryo-EM structures of RAG SEC. \u003cstrong\u003ea\u003c/strong\u003e, A schematic diagram of RAG-mediated DNArecombinationand transposition. SEC is highlighted using deeper colors. \u003cstrong\u003eb\u003c/strong\u003e, Assembly of SEC for cryo-EM study and transposition assays. \u003cstrong\u003ec,d,\u003c/strong\u003e The overall structures of SEC-0 and SEC-PHD. Cryo-EM maps and models are shown as colored surface and cartoons. Map volume for the partial Acidic Hinge (AH) is marked with a black dashed ellipse.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5443361/v1/345eafe9c8bd7d0156fe68c7.png"},{"id":76112354,"identity":"606a0447-a27e-4d36-841b-7b434db2efb4","added_by":"auto","created_at":"2025-02-12 12:10:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":999383,"visible":true,"origin":"","legend":"\u003cp\u003eStructural comparison of Transib and RAG. \u003cstrong\u003ea, \u003c/strong\u003eTop view of SEC-0.\u003cstrong\u003e \u003c/strong\u003eThe region shown in panel b is boxed in dashed black. \u003cstrong\u003eb, \u003c/strong\u003eThe \u003cem\u003etrans\u003c/em\u003e RAG1-RAG2 interactions maintaining the closed conformation in RAG SEC. \u003cstrong\u003ec, \u003c/strong\u003eSuperimposition of Transib TEC and STC. tDNA flanks in Transib TEC and also in RAG SEC (d) are modeled and show as light purple cartoons. The closing motion during TEC-STC transition is indicated with black arrows. \u003cstrong\u003ed, \u003c/strong\u003eSuperimposition of RAG SEC and STC. STC is expanded during SEC-STC transition and indicated with black arrows. \u003cstrong\u003ee, \u003c/strong\u003eStructural changes during Transib-mediated transpositions. The transitions from Transib HFC (PDB: 6PQX) to TEC (PDB: 6PQY) and from TEC to STC (PDB: 6PR5) show overall opening and closing motions. \u003cstrong\u003ef, \u003c/strong\u003eStructural changes during RAG-mediated transpositions. RAG HFC (PDB: 6CG0, NBD domain is omitted in the panel), SEC and STC (PDB: 6OES) overall show the conserved closed form only. Transib helix α9 (e) on ZnB domain and RAG helix O (f) on ZnH2 domain are highlighted in hotpink to show overall conformational changes.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5443361/v1/92d55c1f7ad65c49f11c6603.png"},{"id":76112357,"identity":"465a084a-3c9f-46d9-b2ec-276750306dc1","added_by":"auto","created_at":"2025-02-12 12:10:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1327272,"visible":true,"origin":"","legend":"\u003cp\u003eThe novel PHD-AH binding site on SEC-PHD. \u003cstrong\u003ea\u003c/strong\u003e, Superimposition of SEC-PHD with HFC (PDB: 6CG0) and STC (PDB: 6OES). PHD-AH density is indicated with black arrow, and PHD is shown as hotpink cartoon with molecular surface. RSS coding and tDNA flanks are shown in purple and grey cartoon. \u003cstrong\u003eb, \u003c/strong\u003eTop view of SEC-PHD structure and the corresponding cryo-EM map. Hotpink dashed line shows the trace of AH. The regions shown in panels b and c are boxed in dashed blue and orange, respectively. \u003cstrong\u003ec,d,\u003c/strong\u003e Detailed interactions of PHD and AH with the core RAG. The acidic residues in AH sequence (aa 394-410 of RAG2) are shown in hotpink letters. \u003cstrong\u003ee,\u003c/strong\u003e Structural comparison between RAG1/2 heterodimers with and without PHD binding. Colored cartoons represent the heterodimer with PHD bound and grey cartoon represents the dimer without PHD. The conformational change of helix O and L\u003csub\u003eNO\u003c/sub\u003e upon PHD binding are indicated with black arrows.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5443361/v1/6d355c5bc3d0a734717d748b.png"},{"id":76112358,"identity":"50e874a4-9d68-4511-9f96-d94802426c4e","added_by":"auto","created_at":"2025-02-12 12:10:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":277095,"visible":true,"origin":"","legend":"\u003cp\u003ePHD-AH regulates DNA cleavage and transposition.\u003cstrong\u003e a\u003c/strong\u003e,Superimposition of PHD-H3K4me3 crystal structure (PDB: 2V83) with PHD in SEC-PHD. The region in panel b is outlined in a dashed black box. \u003cstrong\u003eb\u003c/strong\u003e,A zoom-in view of PHD domain. H3K4me3 peptide longer than 6-aa binding to PHD would clash with RAG2 and dislodge PHD from the core RAG. \u003cstrong\u003ec,d,\u003c/strong\u003e Long H3K4me3 peptide (21-aa) rather than short peptide (6-aa) can dislodge PHD from RSS and tDNA binding site and stimulate RSS DNA hairpinning and transposition. Pre-nicked RSSs were used as substares in panel c. \u0026nbsp;\u003cstrong\u003ee,\u003c/strong\u003e Neither long nor short H3K4me3 peptide can stimulate initial RSS DNA binding and nicking when using intact RSS substrates. The results of triplicate assays are averaged and shown.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5443361/v1/267071503f6d68dfe81fda9d.png"},{"id":76112366,"identity":"b134cbee-3796-48da-9f6e-c9e0bbf059c6","added_by":"auto","created_at":"2025-02-12 12:10:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":640943,"visible":true,"origin":"","legend":"\u003cp\u003eThe RAG operating cycle during V(D)J recombination. Core RAG is shown in lime surface, and the core-bound and unbound PHDs are shown as solid and semi-transparent pink ovals, respectively. 12- and 23-RSSs are indicated with yellow and orange triangles. tDNA is shown with purple lines and the clash of two neighboring nucleosomes is indicated. The H3K4me3 peptide tails on nucleosomes are labeled. RAG1 ZnH\u003csub\u003e2\u003c/sub\u003e domain and RAG2 (\u003cem\u003etrans\u003c/em\u003e) are outlined to show the conformational changes from apo-form to HFC (SEC), and RAG stepwise becomes to the low-energy closed form. It is unlikely for SEC to capture a 170° T-form tDNA surrounded by two H3K4me3 nucleosomes. The RAG2 core and non-core domains are able to protect SEC from unwanted transposition and preserve genome stability.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5443361/v1/1be9cac1a0353b7aad36ea02.png"},{"id":88043866,"identity":"2450dfd7-d713-4900-a430-a7be644b2ab3","added_by":"auto","created_at":"2025-07-31 17:47:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4792058,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5443361/v1/9cffab7d-7f68-4a83-889b-b337e2b29b84.pdf"},{"id":76112355,"identity":"53d9ad6c-a850-43f4-858b-11bee9ec3c82","added_by":"auto","created_at":"2025-02-12 12:10:50","extension":"mov","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9874774,"visible":true,"origin":"","legend":"Supplementary Video 1","description":"","filename":"MovieS.mov","url":"https://assets-eu.researchsquare.com/files/rs-5443361/v1/bc7c1e2a7f44415987cd4906.mov"},{"id":76112382,"identity":"d60b77b9-2e9c-456d-a132-fcbbe8118f42","added_by":"auto","created_at":"2025-02-12 12:10:52","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":169313,"visible":true,"origin":"","legend":"Reporting Summary","description":"","filename":"RS.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5443361/v1/fd7e841ef51c683a7c5500bf.pdf"},{"id":76112712,"identity":"1b86878a-e304-4164-a629-5729ac719f13","added_by":"auto","created_at":"2025-02-12 12:18:52","extension":"zip","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":103687066,"visible":true,"origin":"","legend":"9JPU(SEC-PHD)","description":"","filename":"9JPUSECPHD.zip","url":"https://assets-eu.researchsquare.com/files/rs-5443361/v1/d14c68f907728bf49ad737c4.zip"},{"id":76112365,"identity":"6b358d7f-86d4-442b-9dd5-f1bcefb6b911","added_by":"auto","created_at":"2025-02-12 12:10:51","extension":"zip","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":103643638,"visible":true,"origin":"","legend":"9JTU(SEC-1DNA_23RSS)","description":"","filename":"9JTUSEC1DNA23RSS.zip","url":"https://assets-eu.researchsquare.com/files/rs-5443361/v1/f97b86e1257c10e2ad6f3778.zip"},{"id":76112427,"identity":"f7b132ff-9675-46f4-a469-174e378e6b67","added_by":"auto","created_at":"2025-02-12 12:10:55","extension":"zip","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":103276017,"visible":true,"origin":"","legend":"9JPX(SEC-0)","description":"","filename":"9JPXSEC0.zip","url":"https://assets-eu.researchsquare.com/files/rs-5443361/v1/6131012530b55bfa35a5413f.zip"},{"id":76112721,"identity":"e6597652-04cc-44f0-88c8-ba0b2d1c8d73","added_by":"auto","created_at":"2025-02-12 12:18:53","extension":"zip","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":103604464,"visible":true,"origin":"","legend":"9JTS(SEC-1DNA_12RSS)","description":"","filename":"9JTSSEC1DNA12RSS.zip","url":"https://assets-eu.researchsquare.com/files/rs-5443361/v1/6c498b33d1972ed00d373bcb.zip"},{"id":76112406,"identity":"cd729afd-a8f9-4944-9237-b03666300e2d","added_by":"auto","created_at":"2025-02-12 12:10:53","extension":"zip","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":103445394,"visible":true,"origin":"","legend":"9JQN(SEC-2DNA)","description":"","filename":"9JQNSEC2DNA.zip","url":"https://assets-eu.researchsquare.com/files/rs-5443361/v1/979d8fb18c87454ada2f879c.zip"},{"id":76112359,"identity":"a43e904c-b515-40f7-930c-353594248a72","added_by":"auto","created_at":"2025-02-12 12:10:50","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":6630240,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedData.docx","url":"https://assets-eu.researchsquare.com/files/rs-5443361/v1/7621aff6d87a0ed42fa8308e.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"How RAG1/2 evolved from ancestral transposases to initiate V(D)J recombination without transposition","fulltext":[{"header":"Introduction","content":"\u003cp\u003eV(D)J recombination is the essential process generating the adaptive immune system with both diversity and specificity to neutralize a great variety of infectious agents \u003csup\u003e5\u003c/sup\u003e. RAG1/2 (recombination activating genes 1 and 2) protein, a heterotetramer of two RAG1 and two RAG2 subunits, cleaves at the boundaries of RSSs (recombination signal sequences) and flanking V, D and J gene segments of antigen receptors by first nicking and then hairpinning, and the resulting blunt-end signal ends and hairpin-end gene segments (also known as coding flanks) are separately re-joined by Non-Homologous End Joining (NHEJ) (Fig. 1a) \u003csup\u003e1,5,6\u003c/sup\u003e. The core and non-core domains of RAG are respectively responsible for the DNA cleavage and the regulation of V(D)J recombination (Fig. 1b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRAG1 has an RNase H-like (RNH) catalytic core domain (aa 384-1008) \u003csup\u003e6,7\u003c/sup\u003e. Like all RNH-containing transposases, from bacterial Tn5, MuA, Drosophila P element, to eukaryotic Hermes and retroviral integrases \u003csup\u003e8-11\u003c/sup\u003e, RAG1/2 catalytic core not only cleaves at RSS, but also can insert the two cleaved RSS ends as Terminal Inverted Repeats of transposon ends (TIR) into a new 5-bp GC-rich target site \u003cem\u003ein vitro\u003c/em\u003e or \u003cem\u003eex vivo\u003c/em\u003e \u003csup\u003e12,13\u003c/sup\u003e. However, the deleterious transposition by RAG1/2 is suppressed\u003cem\u003e\u0026nbsp;in vivo\u003c/em\u003e \u003csup\u003e14,15\u003c/sup\u003e. RAG2 is absent in transposases including Transib. Beyond its core domain (aa 1-387), the\u0026nbsp;regulatory PHD (aa 410-485) and Acidic Hinge (AH, aa 388-409), which are only present in jawed vertebrates and absent in RAG2L of protoRAG, have been reported to suppress transposition \u003csup\u003e16,17\u003c/sup\u003e. The RAG2 PHD domain is also necessary for recruiting RAG1/2 to V, D and J gene segments on open chromatin by its binding to a histone H3K4me3 tail \u003csup\u003e18\u003c/sup\u003e. Interestingly, the PHD domain also moderately autoinhibits RSS DNA cleavage, and a 21-aa H3K4me3 peptide can release this inhibition \u003csup\u003e3,19\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe mouse apo-RAG1/2 (mRAG) was the first core RAG structure determined \u003csup\u003e20\u003c/sup\u003e. Subsequently, structures of RSS DNA binding, cleavage, and even post-cleavage transposition of RSS into a target DNA (tDNA), have been reported for mRAG, zRAG (zebrafish), Transib, and ProtoRAG \u003csup\u003e7,17,21-27\u003c/sup\u003e. From DNA binding (pre-reaction complex or PRC), nick-forming (NFC) to hairpin-forming complex (HFC), RAG and related recombinases undergo an open-to-close conformational change between its two halves in the DNA cleavage process (Fig. 1a). The same closed conformation is maintained by mRAG in the strand transfer complex (STC), in which the tDNA is kinked twice by 85\u0026deg; (170\u0026deg; overall) 3 bp apart in the 5 bp 5ʹ-CGCCG-3ʹ target site and a further twist out of plane \u003csup\u003e26\u003c/sup\u003e. The severe distortion of tDNA likely hinders RAG-mediated transposition and promotes robust disintegration to revert STC to SEC and tDNA\u003csup\u003e26,27\u003c/sup\u003e. The structure of the core RAG complexed with cleaved RSS signal ends, without coding flanks (SEC), has eluded characterization, and it is unclear if mRAG SEC remains closed or becomes open like Transib bound only to the cleaved TIRs \u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWhen the PHD domain of RAG2 was included in structural studies of V(D)J recombination, a histone H3K4me3 peptide was always added to activate DNA cleavage\u003csup\u003e21,23,25-27\u003c/sup\u003e. As a result, PHD and the other non-core domains of RAG are disordered in all RAG structures reported to date. In the SEC mimic of zRAG in the presence of PHD and H3K4me3, the pre-cleaved RSS DNA occupies the coding flank-binding sites and resembles the HFC structure \u003csup\u003e21\u003c/sup\u003e. How RAG2\u0026rsquo;s PHD inhibits RSS DNA cleavage and transposition remains unknown.\u003c/p\u003e\n\u003cp\u003eHere, we assembled mRAG SEC with the near full-length protein \u0026nbsp;and the pre-cleaved RSS DNAs in the absence of any H3K4me3 peptide, determined the cryo-EM structures at up to 2.95 \u0026Aring; resolutions, and identified a true SEC (SEC-0) and SEC bound by PHD (SEC-PHD) (Fig. 1 and Extended Data Fig. 1). These previously unknown structures illuminate how RAG2 supports the \u0026ldquo;spring-loaded\u0026rdquo; RSS-DNA cleavage and suppresses unwanted transposition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryo-EM analysis of mRAG SEC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mRAG SEC was assembled and purified using size-exclusion chromatography (see Methods). Although the elution profile revealed a single peak. (Extended Data Fig. 2a) and SEC particles on cryo-EM grids appeared homogeneous, the volume surrounding the flank-DNA binding sites (abbreviated as flank binding site below) on 2D averages and initial 3D reconstructions was quite varied, indicating structural heterogeneity \u003csup\u003e21\u003c/sup\u003e (Extended Data Fig. 1). After local 3D classification of the flank binding sites, four major structural species were identified and refined. They are SEC-0, with completely empty flank binding sites (2.95 \u0026Aring;, C2 symmetry), SEC-1DNA, with one blunt-end RSS DNA in one of two flank binding sites (~3.4 \u0026Aring;), SEC-2DNA with two blunt-end DNAs in both sites (3.0 \u0026Aring;, C2 symmetry), and lastly SEC-PHD with a single PHD domain and AH occupying both flank binding sites (3.25 \u0026Aring;, C1 symmetry). The RAG structures in SEC-1DNA and SEC-2DNA are indistinguishable from mRAG HFC and zRAG bound to RSS DNAs \u003csup\u003e21,22\u003c/sup\u003e. For the SEC-PHD structure, the crystal structure of PHD (PDB: 2V83) was readily docked into the large volume (~4 \u0026Aring; resolution) occupying the flank binding site (Fig. 1d), while AH (aa 387-409) was docked as an extended peptide into the remaining volume, but not modeled with individual residues due to the poor resolution of 6 \u0026Aring; or worse (Fig. 1d).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStructure of SEC-0 and implications for recombination and transposition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSEC-0 as well as the other three SEC structures adopts the same closed conformation as HFC and STC, and the RAG1/2 protein chains are well superimposed among them (C\u003csub\u003e\u0026alpha;\u003c/sub\u003e atoms of RAG1 aa 461-1008 and RAG2 aa 1-350, RMSDs \u0026lt; 0.55 \u0026Aring;). The closed conformation of SEC-0 without mediation of the flanking DNAs in HFC or STC is maintained by the RAG1 and RAG2 interactions across the two RAG1/2 heterodimers, which we term \u003cem\u003etrans\u003c/em\u003e interactions. RAG1\u0026rsquo;s helix O and loop L\u003csub\u003eQR\u0026nbsp;\u003c/sub\u003einteract with RAG2\u0026rsquo;s loop L\u003csub\u003eF2F3\u003c/sub\u003e and helix \u0026alpha;\u003csub\u003eE4F1\u0026nbsp;\u003c/sub\u003ein \u003cem\u003etrans\u003c/em\u003e via extensive charge-charge interactions. For example, the K827, R828, K835 and K839 of RAG1 form salt bridges with E341, D334, and D310, and H313 of RAG2 (Fig. 2a and 2b). Even though ProtoRAG2L contains a core region similar to mRAG2, L\u003csub\u003eF2F3\u0026nbsp;\u003c/sub\u003ein RAG2L is much shorter than in mRAG2, and charged residues on L\u003csub\u003eF2F3\u003c/sub\u003e and \u0026alpha;\u003csub\u003eE4F1\u0026nbsp;\u003c/sub\u003eare not conserved. It can be expected that the trans RAG1/2L interactions are absent in ProtoRAG.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRNH-like transposases, most of which are devoid of RAG2-equivalent subunit, can bind, distort and cleave substrate dsDNA independent of a high-energy co-factor. Instead, by a hypothesized \u0026ldquo;spring-loaded\u0026rdquo; mechanism, they may use the initial protein-DNA binding energy to support the subsequent conformational changes and DNA transactions \u003csup\u003e28\u003c/sup\u003e When the apo mRAG1/2 binds to substrate RSS DNAs to form the PRC, RAG1/2 becomes very open, as if a spring is loaded, and the RAG1-RAG2 trans interaction is absent (Supplementary video 1). PRC transforms to NFC by DNA unwinding and protein domain (ZnH2) closing to nick the first DNA strand, then to the most closed HFC to cleave the second DNA strand by forming a hairpin. The SEC-0 with flanking sites empty retains the closed conformation of HFC, indicating that this closed form is the preferred and energy minimum state. Most transposases including Transib accomplish the two-step cleavage reaction without the help of RAG2, but the \u003cem\u003etrans\u003c/em\u003e RAG1-RAG2 interactions and the resulting closed conformation of SEC-0 demonstrate how RAG2 supports and guides RAG1 to accomplish the\u0026nbsp;\u0026ldquo;spring-loaded\u0026rdquo; mechanism in sequential DNA cleavage. While the catalytic residues reside entirely in RAG1, RAG2 becomes an essential accessary subunit to RAG1 by stabilizing RAG1 protein and enabling its \u0026ldquo;spring-loaded\u0026rdquo; mechanism of DNA cleavage\u0026nbsp;\u003csup\u003e20,29,30\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe closed SEC renders transposition by RAG1/2 unlikely because for it to capture a target DNA, the tDNA has to either be already deformed by two 85\u0026deg; kinks 3 bp apart (Extended Data Fig. 4a) or wait for SEC to transiently open with all trans interactions broken. Neither scenario is of high probability. Unlike RAG1/2 recombinase, Transib lacks the RAG2 subunits, and its Transposon End Complex (TEC, equivalent to SEC) is much more open than SEC\u003csup\u003e24\u003c/sup\u003e (Fig. 2c-f and Extended Data Fig. 3). To be captured by Transib, a tDNA would need to be bent by 120\u0026deg; instead of 170\u0026deg; as by mRAG. When Transib carries out transposition, from TEC to STC, it undergoes a 30\u0026deg; closing motion because both Transib subunits bind each flank DNA, and the cleaved tDNA is bent 150\u0026deg;, 30\u0026deg; more than the intact tDNA (Fig. 2c). For RAG1/2, SEC and STC are superimposable (Fig. 2d), and a tDNA needs to be bent 170\u0026deg; before transposition can take place.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA novel PHD-AH binding site revealed by SEC-PHD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the SEC-PHD structure, one PHD domain and the adjacent AH of RAG2 occupy the space vacated by the flank DNAs (Fig. 3a and 3b). The PHD occupies one flank binding site (called \u0026ldquo;RAG1/2 (a)\u0026rdquo;), and the AH fills the other (called \u0026ldquo;RAG1/2(b)\u0026rdquo;). These interactions are mediated by positively charged surfaces of the core RAG1/2 heterodimers and the negatively charged PHD and AH (Fig. 3c, 3d and Extended Data Fig. 2b, 2c). Loop T418-V425 of PHD interacts with the ZnH2 domain of RAG1(a). The side chains of T418 and D424 form hydrogen bonds and a salt bridge with R826 and R734, respectively (Fig. 3c). All backbone carbonyl oxygens except for D424 form polar interactions with R826, K806, R927, K931 and R734. Additionally, S435 of PHD hydrogen bonds with RAG2(a) residues H10 and N11, while N428 interacts with RAG1(a) R848 and M849. V431 interacts with RAG1(a) loop 720-722, and F433 forms a \u0026pi;-cation interaction with RAG2(a) R39 (Fig. 3c). Although the low-resolution map doesn\u0026rsquo;t permit a precise model of AH, aa 394-410 can be confidently located inside the positively charged tunnel lined by K823, R826, K806, R927, K931 and R734 on the RAG1/2(b), suggesting that AH imposes additional hindrance for tDNA binding (Fig. 3d).\u003c/p\u003e\n\u003cp\u003eBecause the PHD domain is wider than a DNA duplex, the flank binding site is remodeled to accommodate it by opening Helix O and loop L\u003csub\u003eNO\u0026nbsp;\u003c/sub\u003e(aa 816-832) in the ZnH2 domain of RAG1(a) (Fig. 3e, Extended Data Fig. 4). In RAG1(a), the peptide between K817 and H818 is flipped, thus changing the conformation of loop L\u003csub\u003eNO\u003c/sub\u003e and pulling it outward, which is accompanied by a 9.7\u0026deg; and 1.6 \u0026Aring; bending of a part of helix O (aa 822-832). In contrast, binding of the AH to the core RAG1 doesn\u0026rsquo;t require any conformational changes, and RAG1(b) is superimposable with SEC-0. Because one of the two PHD domains and its adjacent AH of SEC-0 occupy both flank-DNA binding sites and block tDNA from entering SEC, to eliminate the asymmetric inhibition would require eliminating binding of both PHD domains and linked AHs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRAG2\u0026rsquo;s PHD has been shown to bind an H3K4me3 peptide and release the autoinhibition of RSS DNA cleavage by PHD domains \u003csup\u003e4-6\u003c/sup\u003e. It has been hypothesized that binding of H3K4me3 changes PHD structure and prevents PHD from associating with the core RAG1/2 \u003csup\u003e4\u003c/sup\u003e. To our surprise, PHD domains in SEC-PHD and in the PHD-H3K4me3 complex (PDB: 2V83) are superimposable with an RMSD of 0.687 \u0026Aring; over 58 pairs of C\u003csub\u003e\u0026alpha;\u003c/sub\u003e atoms, and H3K4me3 binding appears to stabilize the otherwise flexible loop aa 471-475 in PHD domain (Fig. 4a, 4b). The buried surface of PHD in the PHD-H3K4me3 complex (735 \u0026Aring;\u003csup\u003e2\u003c/sup\u003e) is smaller than that in SEC-PHD (1,230 \u0026Aring;\u003csup\u003e2\u003c/sup\u003e), and thus H3K4me3 cannot effectively compete with SEC for PHD binding. However, we note that the H3K4me3 peptide bound to PHD in the flank-binding site would clash with the core RAG2, starting from Ala7 of histone H3 onward \u003csup\u003e3,4\u003c/sup\u003e. This suggests a H3K4me3 peptide 6-residue in length would fail to release the inhibition of RSS DNA cleavage and transposition, and only longer peptides, as in nucleosomes, may dislodge and prevent the PHD from occupying the flank binding site.\u003c/p\u003e\n\u003cp\u003eInterestingly, in the apo mRAG structure, the two halves of RAG1/2 core are oriented such that even a single PHD domain cannot fit (Extended Data Fig. 4e). This suggests that PHD domains cannot inhibit initial binding of V, D and J segments to RAG1/2 for RSS cleavage.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInhibition of DNA cleavage and transposition by PHD\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess whether the PHD domain inhibits PRC formation (initial V, D and J substrate binding), DNA nicking, hairpinning, and transposing, and whether long (21 aa) or short (6 aa) H3K4me3 peptides can dislodge and prevent PHD from binding and inhibiting the catalytic activities of core RAG1/2, we performed DNA cleavage and transposition assays with near full-length RAG1/2, and with or without H3K4me3 peptides. Using pre-nicked or pre-cleaved RSSs to compare DNA hairpinning activity or transposition of SEC into supercoiled circular DNA target, respectively, we found that the short peptide had no effect in both reactions; but the long peptide increased the final hairpinning product by less than 2-fold and transposition product by 22-fold (double-ended joint) (Fig. 4c, 4d). First, these results confirm that our hypothesis based on the SEC-PHD structure, and the release of autoinhibition of PHD on the core RAG1/2, depends on the steric clashes between a long methylated H3 tail (such as on a nucleosome) and the RAG1/2 core region. Second, the more than 10-fold different activation levels of the long H3 peptide on hairpinning and transposition likely reflect different autoinhibition mechanisms.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eV, D and J segments are connected to RSS DNA \u003cem\u003ein\u003c/em\u003e \u003cem\u003ecis\u003c/em\u003e, but a tDNA is a separate piece of DNA from RSS and thus \u003cem\u003ein trans\u003c/em\u003e. Moreover, V, D and J segments do not have to bend to bind to RAG1/2 and form the PRC, unlike a tDNA to SEC. Therefore, we used uncut RSS-bordered V and J-like segments to test initial DNA binding and nicking activity. As predicted, no differences were observed among the 3 tested groups, including with or without the 21-aa H3K4me3 (Fig. 4e and Extended Data Fig. 5). In fact, the little to no alteration of initial binding and nicking in the presence of PHD and lack of stimulation by H3K4me3 peptides have been reported previously \u003csup\u003e19,31\u003c/sup\u003e. In this test, we also confirm a less than 2-fold increase of hairpinning product in the presence of the long H3K4me peptide (Extended Data Fig. 5). Indeed, PHD cannot inhibit V, D or J segment binding and nicking of the first strand because binding of RSS DNA results in occupation of flank-binding sites by the coding flanks, and PHD has no chance to compete. A nicked DNA is less rigid than an intact DNA, and thus PHD free of H3K4me3 can interfere with the alignment of nicked DNA substrate for hairpin formation. But the \u003cem\u003ecis\u003c/em\u003e nature of V(D)J recombination renders the inhibitory effect of PHD mild and therefore activation by H3K4me3 rather moderate compared with its effect on transposition. We deduce that acquisition of PHD serves two purposes. One is to recruit RAG1/2 to active and open chromatin domains to increase the substrate specificity of V(D)J recombination, and the second is to inhibit transposition.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWith the SEC structures, we now have a complete presentation of RSS DNA cleavage and post-cleavage DNA transposition by RAG (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Acquisition of the core RAG2 during evolution as exemplified by ProtoRAG2L stabilizes the catalytic subunit. The later addition of the trans interaction as observed between core RAG1/2 subunits increases the \u0026ldquo;spring-loaded\u0026rdquo; DNA cleavage efficiency, and the resulting closed SEC structure presents a barrier to transposition by requiring a tDNA bent nearly 170\u0026deg; with further twist. The AH and PHD addition to the RAG2 core raises the anti-transposition barrier by precluding tDNA binding site in SEC, and the result is inhibition of double-end strand transfer by over 20-fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Remarkably, PHD and AH have no effect on the substrate binding and nicking steps in V(D)J recombination, and only mildly inhibit hairpin formation. RAG1/2 catalyzed processes also depend on transcription and recruitment to open chromatin via the PHD domains (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). A pair of antigen receptor gene segments enabled by the associated nucleosomes can bind to a RAG1/2 recombinase without DNA deformation. But to dislodge two PHD domains alternately bound to an inhibited SEC, a tDNA needs to bring in two nucleosomes, one on each flanking side of the 170\u0026deg; bent target site, and steric clashes of such closely positioned nucleosomes and SEC may further inhibit transposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In short, the evolutionary process of RAG1/2 exemplifies how additional core and non-core domains of RAG2 eliminate unwanted transposition, while making the recombinase more specific and efficient.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e "},{"header":"Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eProtein and DNA preparation\u003c/h2\u003e \u003cp\u003eBoth mouse WT RAG1 (aa 265\u0026ndash;1040) and T490A RAG2 (aa 1-520) were N-terminally tagged by His6-MBP fusion, co-expressed in HEK293T cells and purified as previously described \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. An additional step of Mono Q anion exchange chromatography improved protein purity and removed DNA contamination. The buffer used in amylose affinity purification was 20 mM HEPES (pH 7.4), 500 mM KCl, 5% glycerol, 2 mM DTT, 0.5 mM EDTA. The salt in the protein eluate from the amylose column was diluted to 100 mM before loading onto a Mono Q column (GE Healthcare), which was pre-equilibrated with 20 mM HEPES (pH 7.4), 100 mM KCl, 5% glycerol, 2 mM DTT, 0.5 mM EDTA. mRAG was eluted by a linear gradient of 100\u0026ndash;500 mM KCl. The purified mRAG was buffer-exchanged into storage buffer containing 20 mM HEPES (pH 7.4), 500 mM KCl, 20% glycerol, 0.1 mM EDTA, 2 mM DTT, concentrated to 6\u0026ndash;8 mg/ml, and stored at -80\u0026deg;C. Human HMGB1 (amino acids 1\u0026ndash;163) was prepared as reported previously \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. 12- and 23-RSS DNAs used for structural analyses and biochemical assays (Supplementary table 1) were synthesized as ssDNA and purified using either PAGE or HPLC method (General Biol.). Gel purified oligonucleotides were loaded onto a Glen Gel-Pak column (Glen Research) and eluted in deionized H\u003csub\u003e2\u003c/sub\u003eO. DNA was annealed in an annealing buffer containing 20 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 50 mM NaCl in a Thermocycler.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCryo-EM sample preparation and data collection\u003c/h2\u003e \u003cp\u003eTo prevent catalysis, we incubated WT mRAG, HMGB and DNAs in a Ca\u003csup\u003e2+\u003c/sup\u003e-containing buffer. Both RAG1 and RAG2 subunits contain a N terminal MBP-tag. MBP-mRAG protein, pre-cleaved 12- and 23-RSS signal end DNAs, and HMGB1 (aa 1-163) were mixed at 1:0.9\u0026ndash;1.2:0.9\u0026ndash;1.2:1.8\u0026ndash;2.4 molar ratio in buffer containing 20 mM HEPES (pH 7.4), 100 mM KCl, 5 \u0026micro;M ZnCl\u003csub\u003e2\u003c/sub\u003e, 1 mM DTT, 5% glycerol and 5 mM CaCl\u003csub\u003e2\u003c/sub\u003e and incubated at 37\u0026deg;C for 10 min. The mixture was further purified at 4\u0026deg;C by size exclusion chromatography on a Superdex 200 Increase 10/300 GL column (GE Healthcare) in buffer containing 20 mM HEPES (pH 7.3), 100 mM KCl, 1% glycerol, 1 mM DTT, 5 mM CaCl\u003csub\u003e2\u003c/sub\u003e. The elution peak fractions were pooled and used for cryo-EM grid preparation. 3 \u0026micro;l of the purified SEC (0.3 mg/ml) was spotted on freshly glow-discharged (SuPro Coolglow) QUANTIFOIL R 1.2/1.3 (Cu, 300 mesh) grids at 22\u0026deg;C and blotted for 5 s. The frozen grids were stored in liquid nitrogen before use.\u003c/p\u003e \u003cp\u003eFor structure determination, the frozen grids were loaded into a Titan Krios electron microscope operated at 300 kV for automated image acquisition with SerialEM software, at the Multi-Institute Cryo-EM Facility (MICEF) of NIH. Movies were recorded on a Gatan K2 Summit direct electron detector using the super-resolution mode at 130K nominal magnification (calibrated pixel size of 1.06 \u0026Aring; at the sample level, corresponding to 0.53 \u0026Aring; in super-resolution mode) and defocus values ranging from \u0026minus;\u0026thinsp;0.8 to -3.0 \u0026micro;m. During data collection, the total dose was 70 e\u003csup\u003e\u0026minus;\u003c/sup\u003e/A\u003csup\u003e2\u003c/sup\u003e. The detailed collection statistics are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStructure analysis and model refinement\u003c/h3\u003e\n\u003cp\u003eCryo-EM analysis was performed using CryoSPARC. All frames in each collected movie were aligned and summed using Patch Motion Correction, and CTF estimation were made using Patch CTF Estimation. Blob Picker and Template Picker were used for particle picking, and particles were extracted using a box size of 264 * 264 pixels. 2D classifications and 3D classifications were used to remove junk particles and select the most homogeneous particles for in-depth 3D structural analyses. The final 3D reconstruction for each class was done using Non-Uniform Refinement, and the resulting map was post-processed using DeepEMhancer\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAll reported resolutions are based on the \u0026ldquo;gold standard\u0026rdquo; refinement procedure and the 0.143 Fourier Shell Correlation (FSC) criterion. Local resolution was estimated using Local Resolution Estimation. For model building, STC (PDB: 6OES) and PHD (PDB: 2V83) structures were used as initial models to fit into the maps using Chimera\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, and the resulting models were manually adjusted and rebuilt according to the cryo-EM map in COOT\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Phenix real-space refinement was used to refine the models. The refinement statistics are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The detailed classifications and map qualities of different conformations of SECs are shown in the Supplemental Information (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eDNA cleavage and strand transfer assays\u003c/h3\u003e\n\u003cp\u003eThe RSS DNA cleavage assays were performed in a reaction buffer containing 25 mM HEPES (pH 7.4), 100 mM KCl, 1 mM DTT, 0.1 mg/ml BSA, and 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e. 200 nM each of FAM-labeled 12-RSS and 23-RSS DNAs (including coding flanks, either intact or pre-nicked, shown in Table S1) were incubated with 200 nM of heterotetrametric WT RAG, 400 nM HMGB1 and 1 \u0026micro;M H3K4me3 peptide (Genscript) at 37\u0026deg;C for 0 to 40 min. Reactions were stopped by adding an equal volume of formamide buffer (95% (v/v) formamide, 12 mM EDTA and 0.3% bromophenol blue) and heating at 95\u0026deg;C for 10 min. Cleavage products were separated by 19% TBE-urea PAGE, visualized and quantified using a Typhoon PhosphorImager (GE Healthcare). Plots of biochemical data show the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from three independent experiments using Prism software.\u003c/p\u003e \u003cp\u003eThe strand transfer (transposition) assay was carried out as previously reported\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Briefly, signal-end complex (SEC) was first assembled by mixing WT, pre-cleaved 12- and 23- RSS signal ends without coding flank and HMGB1 at 1:1:1:2 molar ration in a pre-reaction buffer (25 mM HEPES (pH 7.4), 100 mM KCl, 1 mM DTT, and 0.2 mM CaCl\u003csub\u003e2\u003c/sub\u003e) at 37\u0026deg;C for 10 min. The strand transfer rection was carried out by mixing 200 ng supercoiled pUC19 plasmid, 300 nM SEC with 20 \u0026micro;M H3K4me3 peptide in a reaction buffer (25 mM HEPES (pH 7.4), 100 mM KCl, 1 mM DTT, 0.1mg/ml BSA and 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e) and incubating at 37\u0026deg;C for 1 h. The reaction was stopped by adding 25 mM EDTA, and proteins were removed by incubation with 0.4 mg/ml Proteinase K for 30 min at 37\u0026deg;C. DNA products were resuspended in 15 ul loading buffer after ethanol precipitation and separated on a 1.5% agarose gel by electrophoresis. DNA bands were stained with ethidium bromide and quantified using a Typhoon Phosphorimager (GE Healthcare). Data from three independent experiments are averaged and shown with standard deviations using Prism software.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eData and Software Availability\u003c/h2\u003e\n\u003cp\u003eThe accession numbers for the cryo-EM structures and associated density maps of the mouse SEC-0, SEC-PHD, SEC-12DNA, SEC-23DNA, SEC-12/23DNA complexes reported in this paper have been deposited to the PDB and EMDB under accession PDB codes 9JPX, 9JPU, 9JTS, 9JTU and 9JQN, and EMD-61717, EMD-61715, EMD-61816 EMD-61817 and EMD-61730, as specified in Table\u0026nbsp;1.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003e\u0026nbsp;\u003c/h2\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eX.C., M.G. and W.Y. conceived the project; X.C. and L.Y. carried out all experiments and structure determination; H.W. and L.L. helped with cryo-EM data collection; X.C., M.G. and W.Y. supervised the research project; X.C., M.G. and W.Y. prepared the manuscript; All authors participate in the discussions.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe thank the staff at the core facility of Institute of Health Sciences and Technology (Anhui University) for technical support. This research was supported by National Natural Science Foundation of China to X.C. (32371270) and Y.Y. (3220979), Anhui Province Outstanding Youth Fund to X.C. (2308085Y21), Natural Science Research Project of Anhui Educational Committee to X.C. (2022AH030010), and National Institute of Diabetes and Digestive and Kidney Diseases (USA) to M.G. (DK036167) and W.Y. (DK036147).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLiu C, Zhang Y, Liu CC, Schatz DG (2021) Structural insights into the evolution of the RAG recombinase. Nat Rev Immunol\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJones JM, Simkus C (2009) The roles of the RAG1 and RAG2 non-core regions in V(D)J recombination and lymphocyte development. 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Acta Crystallogr D Biol Crystallogr 66:486\u0026ndash;501\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Statistics of cryo-EM data collection and structure refinement\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"width: 780px;\" border=\"1\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 199px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.706px;\"\u003e\n \u003cp\u003eSEC-0\u003c/p\u003e\n \u003cp\u003e(EMDB: EMD-61717)\u003c/p\u003e\n \u003cp\u003e(PDB: 9JPX)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109.294px;\"\u003e\n \u003cp\u003eSEC-PHD\u003c/p\u003e\n \u003cp\u003e(EMDB: EMD-61715)\u003c/p\u003e\n \u003cp\u003e(PDB: 9JPU)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003eSEC-1DNA (12RSS side)\u003c/p\u003e\n \u003cp\u003e(EMDB: EMD-61816)\u003c/p\u003e\n \u003cp\u003e(PDB: 9JTS)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003eSEC-1DNA (23RSS side)\u003c/p\u003e\n \u003cp\u003e(EMDB: EMD-61817)\u003c/p\u003e\n \u003cp\u003e(PDB: 9JTU)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003eSEC-2DNA\u003c/p\u003e\n \u003cp\u003e(EMDB: EMD-61730)\u003c/p\u003e\n \u003cp\u003e(PDB: 9JQN)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 199px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eData collection and processing\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.706px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109.294px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 199px;\"\u003e\n \u003cp\u003eMagnification \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.706px;\"\u003e\n \u003cp\u003e130,000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109.294px;\"\u003e\n \u003cp\u003e130,000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e130,000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e130,000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e130,000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 199px;\"\u003e\n \u003cp\u003eVoltage (kV)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.706px;\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109.294px;\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 199px;\"\u003e\n \u003cp\u003eElectron exposure (e\u003csup\u003e\u0026ndash;\u003c/sup\u003e/\u0026Aring;\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.706px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109.294px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 199px;\"\u003e\n \u003cp\u003eDefocus range (\u0026mu;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.706px;\"\u003e\n \u003cp\u003e-0.8 to -3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109.294px;\"\u003e\n \u003cp\u003e-0.8 to -3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e-0.8 to -3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e-0.8 to -3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e-0.8 to -3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 199px;\"\u003e\n \u003cp\u003ePixel size (\u0026Aring;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.706px;\"\u003e\n \u003cp\u003e1.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109.294px;\"\u003e\n \u003cp\u003e1.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e1.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e1.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e1.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 199px;\"\u003e\n \u003cp\u003eSymmetry imposed\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.706px;\"\u003e\n \u003cp\u003eC2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109.294px;\"\u003e\n \u003cp\u003eC1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003eC1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003eC1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003eC2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 199px;\"\u003e\n \u003cp\u003eInitial particle images (no.)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.706px;\"\u003e\n \u003cp\u003e6,917,387\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109.294px;\"\u003e\n \u003cp\u003e6,917,387\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e6,917,387\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e6,917,387\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e6,917,387\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 199px;\"\u003e\n \u003cp\u003eFinal particle images (no.)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.706px;\"\u003e\n \u003cp\u003e260,975\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109.294px;\"\u003e\n \u003cp\u003e57,418\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e90,752\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e72,732\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e144,322\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 199px;\"\u003e\n \u003cp\u003eMap resolution (\u0026Aring;)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; (FSC threshold=0.143)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.706px;\"\u003e\n \u003cp\u003e2.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109.294px;\"\u003e\n \u003cp\u003e3.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e3.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e3.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e3.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 199px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRefinement\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.706px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109.294px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 199px;\"\u003e\n \u003cp\u003eInitial model used (PDB code)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.706px;\"\u003e\n \u003cp\u003e9JPU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109.294px;\"\u003e\n \u003cp\u003e6OES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e9JPU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e9JPU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e9JPU\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 199px;\"\u003e\n \u003cp\u003eModel resolution (\u0026Aring;)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; (FSC threshold=0.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.706px;\"\u003e\n \u003cp\u003e3.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109.294px;\"\u003e\n \u003cp\u003e3.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e3.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 199px;\"\u003e\n \u003cp\u003eModel composition\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; Non-hydrogen atoms\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; Protein residues\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; Ligands (nucleotide)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.706px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e15,399\u003c/p\u003e\n \u003cp\u003e1,784\u003c/p\u003e\n \u003cp\u003e58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109.294px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e15,963\u003c/p\u003e\n \u003cp\u003e1,853\u003c/p\u003e\n \u003cp\u003e58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e18,710\u003c/p\u003e\n \u003cp\u003e1,928\u003c/p\u003e\n \u003cp\u003e164\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e18,710\u003c/p\u003e\n \u003cp\u003e1,928\u003c/p\u003e\n \u003cp\u003e164\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e16,488\u003c/p\u003e\n \u003cp\u003e1,784\u003c/p\u003e\n \u003cp\u003e112\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 199px;\"\u003e\n \u003cp\u003e\u003cem\u003eB\u003c/em\u003e factors (\u0026Aring;\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; Protein\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; Ligand (nucleotide)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.706px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e64.77\u003c/p\u003e\n \u003cp\u003e77.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109.294px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e77.87\u003c/p\u003e\n \u003cp\u003e96.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e73.18\u003c/p\u003e\n \u003cp\u003e152.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e72.61\u003c/p\u003e\n \u003cp\u003e148.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e56.11\u003c/p\u003e\n \u003cp\u003e93.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 199px;\"\u003e\n \u003cp\u003eR.m.s. deviations\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; Bond lengths (\u0026Aring;)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; Bond angles (\u0026deg;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.706px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.539\u003c/p\u003e\n \u003cp\u003e0.006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109.294px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.471\u003c/p\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.579\u003c/p\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.575\u003c/p\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.587\u003c/p\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 199px;\"\u003e\n \u003cp\u003e\u0026nbsp;Validation\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; MolProbity score\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; Clashscore\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; Rotamers outliers (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.706px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.94\u003c/p\u003e\n \u003cp\u003e6.12\u003c/p\u003e\n \u003cp\u003e2.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109.294px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.78\u003c/p\u003e\n \u003cp\u003e6.39\u003c/p\u003e\n \u003cp\u003e1.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.81\u003c/p\u003e\n \u003cp\u003e10.24\u003c/p\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.85\u003c/p\u003e\n \u003cp\u003e10.41\u003c/p\u003e\n \u003cp\u003e0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.78\u003c/p\u003e\n \u003cp\u003e9.07\u003c/p\u003e\n \u003cp\u003e0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 199px;\"\u003e\n \u003cp\u003e\u0026nbsp;Ramachandran plot\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; Favored (%)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; Allowed (%)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; Disallowed (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.706px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e95.32\u003c/p\u003e\n \u003cp\u003e4.68\u003c/p\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 109.294px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e96.51\u003c/p\u003e\n \u003cp\u003e3.43\u003c/p\u003e\n \u003cp\u003e0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e95.92\u003c/p\u003e\n \u003cp\u003e4.08\u003c/p\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e95.45\u003c/p\u003e\n \u003cp\u003e4.55\u003c/p\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e95.71\u003c/p\u003e\n \u003cp\u003e4.12\u003c/p\u003e\n \u003cp\u003e0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"RAG, transposition, target DNA, PHD, H3K4me3","lastPublishedDoi":"10.21203/rs.3.rs-5443361/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5443361/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe RAG1/2 recombinase, which initiates V(D)J recombination in jawed vertebrates, evolved from RNaseH-like transposases such as Transib and ProtoRAG \u003csup\u003e1\u003c/sup\u003e. However, its post-cleavage transposase activity is strictly suppressed. Previous structural studies have focused only on the conserved core domains of RAG1/2, leaving the regulatory mechanisms of the non-core regions unclear. To investigate how RAG1/2 suppresses transposition and regulates DNA cleavage, we determined cryo-EM structures of nearly full-length RAG1/2 complexed with cleaved Recombination Signal Sequences (RSS) in a Signal-End Complex (SEC), at resolutions up to 2.95 Å. Two key structures, SEC-0 and SEC-PHD, reveal distinct regulatory roles of RAG2, which is absent in Transib transposase. SEC-0 displays a closed conformation, revealing that the core RAG2 facilitates sequential DNA cleavage by stabilizing the RSS-cleaved states in a \"spring-loaded\" mechanism. SEC-PHD reveals how RAG2’s non-core PHD and Acidic Hinge (AH) domains, which are absent in ProtoRAG, inhibit target DNA binding in transposition. Histone H3K4me3, which recruits RAG1/2 to RSS sites, does not influence RAG1/2 binding to V, D or J gene segments bordered by RSS \u003csup\u003e2\u003c/sup\u003e. In contrast, the suppressed transposition can be activated by H3K4me3 peptides that dislodge the inhibitory PHD domain \u003csup\u003e3,4\u003c/sup\u003e. To achieve this de-repression in vivo, however, would require an unlikely close placement of two nucleosomes flanking a target DNA bent by nearly 180°. Our structural and biochemical results elucidate how RAG1 has acquired RAG2 and utilizes its core and non-core domains to enhance V(D)J recombination and suppress transposition.\u003c/p\u003e","manuscriptTitle":"How RAG1/2 evolved from ancestral transposases to initiate V(D)J recombination without transposition","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-12 12:10:41","doi":"10.21203/rs.3.rs-5443361/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6a0cb417-b0ac-4a37-b38f-043f09f50de8","owner":[],"postedDate":"February 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":43077422,"name":"Biological sciences/Biochemistry/Structural biology/Electron microscopy/Cryoelectron microscopy"},{"id":43077423,"name":"Biological sciences/Molecular biology/DNA recombination"},{"id":43077424,"name":"Biological sciences/Biochemistry/Enzymes/Recombinases"}],"tags":[],"updatedAt":"2025-07-31T17:47:44+00:00","versionOfRecord":{"articleIdentity":"rs-5443361","link":"https://doi.org/10.1073/pnas.2512362122","journal":{"identity":"proceedings-of-the-national-academy-of-sciences","isVorOnly":true,"title":"Proceedings of the National Academy of Sciences"},"publishedOn":"2025-07-29 00:00:00","publishedOnDateReadable":"July 29th, 2025"},"versionCreatedAt":"2025-02-12 12:10:41","video":"","vorDoi":"10.1073/pnas.2512362122","vorDoiUrl":"https://doi.org/10.1073/pnas.2512362122","workflowStages":[]},"version":"v1","identity":"rs-5443361","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5443361","identity":"rs-5443361","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}