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Structural basis of long-range to short-range synaptic transition in NHEJ

Abstract

DNA double-strand breaks (DSBs) are a highly cytotoxic form of DNA damage and the incorrect repair of DSBs is linked to carcinogenesis1,2. The conserved error-prone non-homologous end joining (NHEJ) pathway has a key role in determining the effects of DSB-inducing agents that are used to treat cancer as well as the generation of the diversity in antibodies and T cell receptors2,3. Here we applied single-particle cryo-electron microscopy to visualize two key DNA–protein complexes that are formed by human NHEJ factors. The Ku70/80 heterodimer (Ku), the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), DNA ligase IV (LigIV), XRCC4 and XLF form a long-range synaptic complex, in which the DNA ends are held approximately 115 Å apart. Two DNA end-bound subcomplexes comprising Ku and DNA-PKcs are linked by interactions between the DNA-PKcs subunits and a scaffold comprising LigIV, XRCC4, XLF, XRCC4 and LigIV. The relative orientation of the DNA-PKcs molecules suggests a mechanism for autophosphorylation in trans, which leads to the dissociation of DNA-PKcs and the transition into the short-range synaptic complex. Within this complex, the Ku-bound DNA ends are aligned for processing and ligation by the XLF-anchored scaffold, and a single catalytic domain of LigIV is stably associated with a nick between the two Ku molecules, which suggests that the joining of both strands of a DSB involves both LigIV molecules.

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Fig. 1: Cryo-electron microscopy structure of the LR synaptic complex.
Fig. 2: Activation mechanism of DNA-PKcs in the LR synaptic complex.
Fig. 3: Cryo-electron microscopy structure of the SR synaptic complex.
Fig. 4: Structural transition from the LR to the SR synaptic complex.

Data availability

Cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-23510 (overall LR complex), EMD-23511 (DNA-PK–N-BRCT in the LR complex), EMD-23512 (LigIV–XRCC4–XLF–XRCC4–LigIV in the LR complex), EMD-23509 (overall SR complex), EMD-23513 (distal Ku–LigIV catalytic domain in the SR complex), EMD-23514 (proximal Ku in the SR complex) and EMD-23515 (LigIV–XRCC4–XLF–XRCC4–LigIV in the SR complex). Model coordinates have been deposited in the Protein Data Bank under accession numbers 7LT3 (the LR complex) and 7LSY (the SR complex).

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Acknowledgements

We thank J. Pattie for computer support, J. Meyers, R. M. Haynes and H. Scott at the PNCC for data collection support, D. Ramsden for the gift of a rabbit anti-phosphoT2609 reagent, A. Rosenzweig and I. Radhakrishnan for discussion and comments on the manuscript. This work was supported by a Cornew Innovation Award from the Chemistry of Life Processes Institute at Northwestern University (to Y.H.), a Catalyst Award by the Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust (to Y.H.), an Institutional Research Grant from the American Cancer Society (IRG-15-173-21 to Y.H.), an H Foundation Core Facility Pilot Project Award (to Y.H.), a Pilot Project Award under U54CA193419 (to Y.H.) and NIH grant R01 GM135651 (to Y.H.). S.C. is supported by the Molecular Biophysics Training Program from NIGMS/NIH (5T32 GM008382). A portion of this research was supported by NIH grant U24GM129547 and performed at the PNCC at OHSU and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. This work used the Sapphire imager from the Northwestern University Keck Biophysics Facility funded by NIH grant 1S10OD026963-01, as well as the resources of the Northwestern University Structural Biology Facility, which is generously supported by NCI CCSG P30 CA060553 grant awarded to the Robert H. Lurie Comprehensive Cancer Center. The Gatan K2 direct electron detector was purchased with funds provided by the Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust. Work in the Lees-Miller laboratory was supported by Canadian Institutes of Health grant 16939 and the Engineered Air Chair in Cancer Research. Work in the Tomkinson laboratory was supported by NIH grant R01GM047251, as well as the University of New Mexico Comprehensive Cancer Center supported by NCI CCSG P30 CA118100 grant. The collaboration between the He, Lees-Miller and Tomkinson laboratories was supported by NCI P01 CA092584.

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Authors and Affiliations

Authors

Contributions

Y.H., S.P.L.-M. and A.E.T. conceived the project. S.C. performed most of the experiments and collected and analysed cryo-EM data with Y.H. L.L. and T.N. contributed to protein purification. S.C. built the models with help from Y.H. S.C., S.F., A.W. and Y.H. wrote the manuscript with input from all other authors.

Corresponding author

Correspondence to Yuan He.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Optimization of the LR synaptic complex assembly with various DNA substrates.

a, Schematic showing the Y35 blunt-end DNA substrate. Complex assembly was attempted (supplying DNA-PKcs, Ku, XLF and LigIV–XRCC4) before purification by RNase H elution. b, A representative negative-staining raw micrograph of the complex assembled as described in a. The raw micrograph is representative of 24 micrographs. c, Representative two-dimensional class averages of the complex assembled as described in a, showing the appearance of only the DNA-PK complex despite the addition of XLF and LigIV–XRCC4. df, Same procedure as ac, showing the complex assembly with the same Y35 substrate, but adding XLF and LigIV–XRCC4 to the purified DNA-PK complex after RNase H elution. The raw micrograph is representative of 27 micrographs. In f, the two-dimensional class averages representing the characteristic view of scarce but existing LR complex were obtained. gi, Same procedure as ac, showing the complex assembly using Y30–T40–c8 DNA substrate with 40 nt flexible poly(T) and 8 bp of complementary ends as the 3′ overhang. Although the single-stranded poly(T) overhang and the 8-bp complementary region contribute to the stability of the complex, they are not observed in any part of the reconstructed density map, presumably because these single-strand DNA tethers are too flexible to be aligned with the rest of the complex. The raw micrograph is representative of 24 micrographs. The complex was assembled before RNase H elution as described in a. In i, the majority of the two-dimensional classes correspond to the LR complex. j, A representative cryo-EM raw micrograph (out of 17,114 micrographs in total) of the LR complex assembled with the Y30–T40–c8 DNA substrate shown in g. k, Representative two-dimensional class averages of particles (329,784 in total) contributing to the final reconstruction of the LR complex. l, Silver-stained SDS–PAGE (4–12% gradient, biologically replicated three times) showing the input purified subunits (Ku, DNA-PKcs, LigIV–XRCC4, and XLF) and the RNase H purified LR and SR complex for cryo-EM data collection. All representative micrographs in b, e, h, j are from at least three biologically replicated experiments. For gel source data, see Supplementary Fig. 1. m, Protein–protein interaction network between the components of the LR complex. Major unmodelled regions are shown in grey. Well-documented hetero- or homo-dimers are grouped by red dashed lines. Alternative protein–protein interactions are depicted by black dashed lines. The globular domain within the Ku80 C-terminal region (CTR) is completely flexible in the LR complex, and we do not see evidence of the Ku80 CTR domain swap that was previously observed30. The putative distance between one Ku80 CTR globular domain and the other copy of the Ku80 C-terminal helix is too far to be reached by the 18-amino acid linker within Ku80. BRCTs, tandem BRCT domains; CC, coiled-coil domain; CTD, C-terminal domain; HD, head domain; KD, kinase domain; M-HEAT, middle HEAT domain; N-HEAT, N-terminal HEAT domain; NTD, N-terminal domain; OBD, OB-fold domain.

Extended Data Fig. 2 Data-processing scheme of the LR synaptic complex sample.

a, Flow chart of the cryo-EM data processing procedure. The gold-standard Fourier shell correlation (FSC) curves (0.143 cut-off) show the final resolution of the holo-complex and each body. b, sample maps and fitted models of DNA-PKcs (olive) and dsDNA substrate (cyan) from the LR complex are shown at 4.1 Å resolution. Maps are shown as transparent surfaces and models are shown as sticks.

Extended Data Fig. 3 Comparing the structure of Ku among the LR synaptic complex, the SR synaptic complex and previously published models.

a, Ku structure in the LR complex showing outward rotations of both Ku70 and Ku80 vWA domains. b, Crystal structure of the XLF Ku-binding motif (KBM) bound to Ku showing the outward rotation of only the Ku80 vWA domain29. c, Crystal structure of apo Ku showing no rotation of either Ku70 or Ku80 vWA domains22. d, Conformation of Ku shown in the cryo-EM structure of the apo DNA-PK complex30. The Ku70 vWA domain is rotated outward, triggered by binding of DNA-PKcs. e, f, Two copies of the XLF Ku-binding motif bound to Ku in the SR complex. The conformation of both copies is the same as the one in the LR complex (a), despite the fact that DNA-PKcs is not present. Colour codes for Ku70 and Ku80 are the same as in Fig. 1.

Extended Data Fig. 4 Comparing the structure of the LigIV–XRCC4–XLF scaffold among the LR synaptic complex, the SR synaptic complex and previously published models.

a, Structure of XRCC4–XLF from the LR complex is shown in comparison to the XRCC4–XLF filamentous repeat crystal structures and the ones from the SR complex (both copies). The XLF dimer is used to align all of the models shown here. Solid lines are aligned with the coiled-coils of XLF (vertical) and XRCC4 (tilted), and the angles in between are shown. Dashed lines are aligned with the C-terminal half of the coiled-coil domain of XRCC4 when full helices are present, and the bending angles are also shown. b, XRCC4 in the crystal structure of the human and yeast LigIV–XRCC4 complexes24,34 are shown after aligning with XRCC4 in the LR complex shown in a. The bending of the coiled-coil domain of XRCC4 is more similar to the one in the SR than in the LR complex. c, Structure of the LigIV–XRCC4 complex from the LR complex is shown in comparison to human and yeast LigIV–XRCC4 crystal structures and ones from the SR complex (both copies). Colour codes for XLF, XRCC4 and LigIV BRCT domains are the same as in Fig. 1.

Extended Data Fig. 5 Surface electrostatic potential and conservation of different areas in the LR synaptic complex.

a, Magnified view of the interaction surface between LigIV N-BRCT domain and Ku70 vWA domains coloured by sequence conservation. b, Magnified view of the DNA-PKcs–DNA-PKcs interaction surface coloured by sequence conservation. c, Surface electrostatic potential view of DNA-PKcs near its FAT domain, showing its negatively charged interface with the XRCC4 C-terminal region (ribbon). The approximate path of the XRCC4 C-terminal peptide containing multiple phosphorylation sites is depicted. The sphere depicts the location of a cancer-associated truncation mutation that occurs at the interface. d, Surface electrostatic potential view of DNA-PKcs DEB and DEB-A helix. The DNA-interaction surface is positively charged. When models are not coloured by either surface electrostatic potential or sequence conservation, the colour codes are the same as in Fig. 1. We cannot rule out the unlikely possibility that the stabilization of the DEB helix is due to the presence of a 3′ overhang that existed in our DNA substrate design (Extended Data Fig. 1g).

Extended Data Fig. 6 Comparing the dimerization of DNA-PKcs in the LR synaptic complex with other PIKK family dimers.

a, Structure of the two DNA-PKcs in the LR complex. The kinase domain is aligned with the homologous domains in b and c as an anchor point. b, c, Dimer of ATR–ATRIP (b) and ATM (c) showing the aligned kinase domain and corresponding N-HEAT regions in the aligned copy. The symmetric-look front views are shown at the bottom left corner. Each protomer of ATR–ATRIP and ATM is coloured the same as the corresponding DNA-PKcs protomer, in olive (the aligned copy) and dark khaki (the other copy). d, Domain organization of DNA-PKcs compared with ATR and ATM. Abbreviations are as in Fig. 1. In our model, both the ABCDE (T2609, S2612, T2620, S2624, T2638 and T2647) and the PQR (S2023, S2029, S2041, S2053 and S2056) phosphorylation sites are located within disordered loops of DNA-PKcs 2606–2720 and 1993–2084, respectively (Fig. 2b, d). The kinase active centre from the opposite side cannot reach most of the ABCDE sites unless the YRPD-interaction (YRPD-I) loop (residues 2586–2604) is peeled off from the YRPD motif (Fig. 2d). In turn, this conformational change potentially disrupts the DNA-PKcs–DNA-PKcs dimerization interface through loop 2569–2585 (Fig. 1d). Similarly, some PQR sites are located too far from the trans kinase active centre. PQR-autophosphorylation-induced changes could have a direct effect on the Ku80 CTR–DNA-PKcs interface at the bottom of the cradle (Fig. 2d), potentially inducing the domain swap of Ku80 that was previously observed30.

Extended Data Fig. 7 Optimization of the SR synaptic complex assembly with various DNA substrates.

a, Schematic showing the Y30–c4 DNA substrate with 4-nt 3′ complementary overhang. The complex was assembled before RNase H elution. b, A representative negative-staining raw micrograph of the complex assembled as described in a. The raw micrograph is representative of 23 micrographs. c, Representative two-dimensional class averages of the complex assembled as described in a. d, Cryo-EM map reconstructed from a small dataset using Y30–c4 DNA substrate shown in a. The map is coloured according to the estimation of the local resolution. eh, Same procedure as ad, showing the complex assembly with the Y30 blunt-end substrate. Stably assembled SR complexes on DNA substrates with either complementary or blunt ends indicates that these complexes are stable in the absence of any bridging effect from DNA. The raw micrograph is representative of 24 micrographs. il, Same procedure as ad, showing the complex assembly with the Y14–T2–c20–n10–10 substrate, with one central single non-ligatable nick. The raw micrograph is representative of 24 micrographs. Strand e is added last after mixing the two halves together with NHEJ factors. m, A representative cryo-EM raw micrograph (out of 32,723 total images) of the SR complex assembled with the Y14–T2–c20–n10–10 DNA substrate shown in i. n, Representative two-dimensional class averages of particles (175,866 in total) contributing to the final reconstruction of the SR complex. All representative micrographs in b, f, j, m are from at least three biologically replicated experiments. o, Protein–protein interaction network between the components of the SR complex. Major unmodelled regions are shown in grey. Well-documented hetero- or homo-dimers are grouped by red dashed circles. Alternative protein–protein interactions are depicted by black dashed lines.

Extended Data Fig. 8 Data-processing scheme of the SR synaptic complex sample.

Flow chart of the cryo-EM data processing procedure. The gold-standard Fourier shell correlation curves (0.143 cut-off) show the final resolution of the holo-complex and each body.

Extended Data Fig. 9 Surface conservation of different areas in the SR synaptic complex.

a, Magnified view of the interaction surface between the LigIV DBD and Ku70 vWA domain coloured by sequence conservation. DNA-PKcs clashes with LigIV DBD when Ku is aligned between the LR and SR complex. b, Magnified view of the coiled-coil domain of XLF at its C-terminal tip coloured by sequence conservation. When models are not coloured by sequence conservation, the colour codes are the same as in Fig. 3. c, Superimposition of two asymmetric SR complexes after a 180° flip shown in front (top) and top (bottom) views. The XLF homodimer is used for aligning the two conformers. LigIV catalytic domains are hidden for clarity purposes. The transition from the apo state to the flipped state indicates potential conformational changes during the tandem ligation. Paths of DNA are also highlighted by dashed lines. d, Magnified view showing the relative positions of the two off-centred nicks between the two conformers. The two preferential nick positions are separated by approximately 4 bp. Consistently, dsDNA with 4-nt 3′ overhang, a major end-processing product of the NHEJ nuclease Artemis78, is reported to be a favoured substrate for NHEJ50. Our model suggests that dsDNA with a 4-nt 3′ overhang will experience minimum DNA translocation to accommodate the two ligation steps (Supplementary Video 3).

Extended Data Fig. 10 Both LR and SR synaptic complexes are able to perform double ligation during NHEJ in vitro.

a, Substrate design for the ligation assay. An internal Cy5 label is added to only the right half of the substrate to visualize the ligation products. A 4-nt 3′ complementary overhang has been introduced on both sides of the substrate. b, Denaturing gel analysis of end joining by the LR complex. The reaction comprised 100 nM of DNA, 200 nM of DNA-PKcs and Ku70/80, 500 nM of XLF and 70 nM of X4L4. The asterisk indicates an alternative secondary structure or impurity of the Cy5-labelled oligomer. The size of the DNA substrates and ligation products are labelled on the left (unit: bp). c, Denaturing gel analysis of end joining by the SR complex. The final factor concentrations are the same as in b. For gel source data, see Supplementary Fig. 1. Similar conditions for the gels have been replicated as biological replicates twice.

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-2 and Supplementary Figures 1-7.

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Video 1

Density map and model fitting of the Long-range synaptic complex.

Video 2

Density map and model fitting of the Short-range synaptic complex.

Video 3

Model showing the conformational changes within the Short-range synaptic complex during the tandem ligation of two dsDNA nicks. DNA substrate with 4nt 3’ overhang is used as the example.

Video 4

Model showing the conformational changes during the transition from the Long-range to the Short-range synaptic state.

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Chen, S., Lee, L., Naila, T. et al. Structural basis of long-range to short-range synaptic transition in NHEJ. Nature 593, 294–298 (2021). https://doi.org/10.1038/s41586-021-03458-7

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