Abstract
In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70–Ku80 heterodimer (Ku), X-ray repair cross complementing 4 (XRCC4) in complex with DNA ligase 4 (X4L4) and XRCC4-like factor (XLF) form a flexible scaffold that holds the broken DNA ends together. Insights into the architectural organization of the NHEJ scaffold and its regulation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were recently obtained by single-particle cryo-electron microscopy analysis. However, several regions, especially the C-terminal regions (CTRs) of the XRCC4 and XLF scaffolding proteins, have largely remained unresolved in experimental structures, which hampers the understanding of their functions. Here we used magnetic resonance techniques and biochemical assays to comprehensively characterize the interactions and dynamics of the XRCC4 and XLF CTRs at residue resolution. We show that the CTRs of XRCC4 and XLF are intrinsically disordered and form a network of multivalent heterotypic and homotypic interactions that promotes robust cellular NHEJ activity. Importantly, we demonstrate that the multivalent interactions of these CTRs lead to the formation of XLF and X4L4 condensates in vitro, which can recruit relevant effectors and critically stimulate DNA end ligation. Our work highlights the role of disordered regions in the mechanism and dynamics of NHEJ and lays the groundwork for the investigation of NHEJ protein disorder and its associated condensates inside cells with implications in cancer biology, immunology and the development of genome-editing strategies.
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Data availability
The NMR assignments for the CTRs of XLF, XRCC4 and BRCT1 can be accessed from the Biological Magnetic Resonance Data Bank (BMRB) under accession codes 52387, 52398 and 52399, respectively. The SAXS data for XLF, XRCC4 in complex with BRCTs and the CTR of XRCC4 can be accessed from the Small-Angle Scattering Biological Data Bank (SASBDB) under accession codes SASDU47, SASDU57 and SASDU67, respectively. The previously published structures of proteins were retrieved from the Protein Data Bank (PDB) under accession codes 2R9A (XLF) and 7LSY (NHEJ short-range synapsis). All data supporting the findings are provided within the paper and the Supplementary Information. Other materials generated in this work will be made available upon request. Source data are provided with this paper.
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Acknowledgements
We acknowledge SOLEIL for provision of synchrotron radiation facilities and we thank A. Thureau for assistance in using beamline SWING. We thank K. Bugge, B. B. Kragelund and J. G. Olsen for accessing the DL-SAXS facility at the Diamond Light Source (grant no. EP/R042683/1), N. Salvi and N. Bolik-Coulon for fruitful discussions and K. Meek and E. Rothenberg for critical reading of the paper. This work was funded by the French National Research Agency (ANR-18-CE29-0003 NANO-DISPRO, ANR-17-CE2-0020 NHEJLIG4 and ANR-20-CE11-0026-04 BREAKDANCE) and French Ligue against Cancer (équipe labellisée). This work was supported by the Fondation ARC pour la recherche sur le cancer (grant no. ARCDOC42021120004347), the ERC (grant agreement 279519 (2F4BIODYN) to F.F. and 835161 (DynamicAssemblies) to M.B.) and Equipex (contract ANR-10-EQPX-09, Paris en resonance). A.K.A. was supported by grant R35-GM131780 from the National Institutes of Health (NIH). J.M.S. was supported by grant R01CA256989 from the NIH. M.C.A. was supported by grant F99CA284248 from the NIH. Financial support from the IR INFRANALYTICS FR2054 CNRS for conducting the research is gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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F.F., M.M., D.D.V, V.B. and M.Bl. conceptualized the project and obtained the main funding. D.D.V. prepared reagents and performed the NMR and in vitro experiments with the help of P.P., Z.W., L.C., G.B. and Z.G. M.Br., M.M., M.C.-A. and J.M.S. prepared reagents and performed the cellular experiments. A.B. and V.B. conducted the EPR experiments. M.Bl., J.M.S., Z.G., P.C., P.P., L.C., G.B. and A.K.A. assisted in conceptualization and analysis of data. D.D.V., M.M. and F.F. wrote the paper and all coauthors edited and approved the final version of the paper.
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Extended data
Extended Data Fig. 1 Effects of XLF mutations on No Indel EJ with endogenous XRCC4.
(Left) GFP frequencies are normalized to parallel GFP transfections to account for transfection efficiency. n = 9 biologically independent transfections. Statistical significance was established with unpaired two-tailed t-test with Holm-Sidak correction. ****P < 0.0001, n.s. = not significant, the exact P-values from top to bottom are < 0.0001, 0.8252, < 0.0001, 0.4748, and 0.0782, respectively, #x represents fold effect. (Right) Immunoblots show levels of XRCC4, XLF-WT, and XLF mutants, data are represented as mean values ± SD.
Extended Data Fig. 2 NMR parameters of XLF.
Only the peaks belonging to the residues in the CTR are visible and analyzed. (a) Heteronuclear [1H]-15N Overhauser effects (hNOE) of XLF. (b) CPMG transverse relaxation rate (R2), (c) longitudinal relaxation rate (R1), (d) R2/R1 ratio of XLF and (e) secondary structure propensity of XLF calculated using Cα and Cβ chemical shift. All parameters were measured on an 800 MHz spectrometer at 298 K; the residues in the DNA-binding motif are highlighted in blue; the residues in the Ku-binding motif are highlighted in gray. The error bar represents the standard error with one sigma uncertainty, estimated either from noise propagation (a) or the covariance matrix (b-c) with n=1 independent experiment for each spectrum.
Extended Data Fig. 3 Titration of XLF with DNA monitored by NMR.
Only the peaks belonging to the residues in the CTR are visible and analyzed. (a) Overlay 1H-15N HSQC spectra of XLF (red) after adding 0.1, 0.5, 0.85, 1.25, 2, and 4 equivalents of five types of DNA as indicated. (b) Top: Chemical shift perturbation of XLF before and after adding 4 equivalents of different types of DNA; bottom: as in the top figure but the binding site is enlarged. (c) The chemical shift perturbation of the most affected residues is presented as a function of the amounts of DNA added during the titration experiments, with the color scheme similar to (b). (d) The apparent affinity of XLF (\({{{{{\rm{K}}}}}^{{{{\rm{D}}}}}}_{{{{\rm{app}}}}}\pm {{{\rm{SD}}}}\)) for DNA by fitting 2D line shape of affected peaks by TITAN software. Upon saturation of XLF with DNA, the peaks corresponding to the bound residues remain rather sharp across varying DNA lengths and compositions. Notably, these peaks persist within the chemical shift range of the disordered region (7.5-8.5 ppm, 1H chemical shift). This observation suggests that the DNA-binding domain of XLF remains dynamic even after binding to DNA. The fitted parameters from TITAN are provided in the source data. (e) Electrophoretic mobility shift assay (EMSA) of different XLF constructs with DNA (Smal-linearized pUC19), the experiments were carried out as duplicates with similar result.
Extended Data Fig. 4 XLFCTR interacts with the folded domain.
Only the peaks belonging to the residues in the CTR are visible and analyzed. (a) Transverse relaxation rate (R2) of XLFCTR (green) after adding 1 (orange) or 2 (pink) equivalents of XLF1-224 compared with full-length XLF (red). (b) R2 of XLF in comparison with the three mutants in the DNA-binding motif and the Ku-binding motif. All the rates were measured on an 800 MHz spectrometer at 298 K, the residues in the DNA-binding motif are highlighted in blue, and the residues in the Ku-binding motif are highlighted in gray. (c)15N R2 values of the most affected residues in XLFCTR plotted against the concentration of XLF1-224 at a constant XLFCTR concentration of 250 μM. (d) Estimation of the apparent residue-specific dissociation constants from the in trans titration. The KD values (bar plot, black) are extracted from the slopes of the R2 vs. concentration in XLF1-224 plotted in (c).The in trans residue-specific apparent dissociation constants KD and the R2 of the residues in full-length XLF were then used to calculate the percentage of the bound form of the CTR in the full-length XLF (scatter plot, red solid circles). The error bar represents the standard error with one sigma uncertainty, estimated either from the covariance matrix (a-c) or a 10000-step Monte Carlo simulation (d) with n=1 independent experiment for each spectrum.
Extended Data Fig. 5 Identifying the region of BRCT1 that interact with XLFCTR.
(a)1H-15N HSQC spectra of BRCT1 (residues 654-759) with resonance assignment. (b) Overlay 1H-15N HSQC spectra of BRCT1 in the absence (orange) and in the presence (cyan) of two equivalents of XLFCTR. (c) top: normalized peak intensity ratios extracted from 1H-15N HSQC spectra of 15N BRCT spectra before (I∘) and after adding 1 (green) or 2 (cyan) equivalents of XLFCTR (I), bottom: chemical shift perturbation (CSP) of BRCT1 after adding 2 equivalents of XLFCTR. (d) BRCT1-XLFCTR complex predicted using AlphaFold-Multimer.
Extended Data Fig. 6 The effects of additive components on LLPS of XLF and X4L4.
Fluorescence microscopy images of X4L4 (1% Cy3 labelling) and XLF with increasing concentrations of (a) Ficoll 400, (b) NaCl or (c) Hexane-1,6-diol. The concentration of X4L4 and XLF was fixed at 10 μM. The scale bar is 16 μm, the experiments were carried out as duplicates with similar results.
Extended Data Fig. 7 The partial FRAP experiments of X4L4-XLF droplets.
(a) The fluorescence images of a representative droplet at various time points during the partial FRAP experiment, with the red box indicating the bleaching zone and the white line representing pixels used to construct the kymograph on the left, the scale bar is 6 μm (b) The fluorescence intensity of the bleached and non-bleached halves during the FRAP experiments, n=4, the concentration of XLF and X4L4 was fixed at 20 μM with 1% fluorescein-labelling XLF. Data are represented as mean values ± SD.
Extended Data Fig. 8 The CTR of XLF is important for promoting LLPS with X4-BRTCs.
(a) Fluorescence microscopy images of XRCC4-BRCTs with increasing concentrations of different XLF mutants. XLF variants were titrated into 10 μM 1% Cy3 labelling X4-BRCTs, the scale bar is 16 μm. (b) Quantification of droplet area percentage of the different XLF constructs in (a). Statistical significance was established with an unpaired two-tailed t-test, *P < 0.05, ns = not significant, the exact P-values from left to right, respectively, are 0.5083, 0.0783, 0.0156, 0.0448,0.1209, 0.0448. Data are represented as mean values ± SD with n=2 independent experiments for the conditions without droplets and for conditions with droplets, n is between 3 and 5 independent experiments. Please refer to the source data file for the exact number of experiments conducted for each condition and construct.
Extended Data Fig. 9 The ability of different XRCC4 and LIG4 constructs undergo LLPS with XLF.
(a) Fluorescence microscopy images of XLF (1% Fluorescein-labelled) with increasing concentrations of different XRCC4 and LIG4 constructs. XRCC4 and LIG4 variants were titrated into 10 μM (monomer) 1% Fluorescein-labelled XLF, the scale bar is 16 μm. (b) Quantification of droplet area percentage of the different X4L4 constructs in (a), for XRCC4, X4-XID and X41-203-BRCTs, the constructs formed aggregates and were not quantified (NA). Statistics with unpaired two-tailed t-test. ****P < 0.0001, ***P < 0.001, **P < 0.01, ns = not significant, the exact P-values from left to right respectively are 0.0067, 0.0021, 0.0011, 1e-8, 9e-8, 4e-6. Data are represented as mean values ± SD with n=2 independent experiments for the conditions without droplets and for conditions with droplets, n is between 5 and 8 independent experiments. Please refer to the source data file for the exact number of experiments conducted for each condition and construct.
Extended Data Fig. 10 In vitro DNA ligation by various X4L4 and XLF constructs.
(a) The image of an agarose gel after fractionation by electrophoresis and SYBR Green I staining of deproteinized cohesive end ligation products from reactions containing X4L4 with different XLF mutants as indicated in the presence and the absence of 4% of PEG8000 at three different time points. The substrate was 2.7 kb XbaI-linearized pUC19 plasmid. The protein concentration is fixed at 2 μM. (b) The quantification of the ligated DNA products of the reactions shown in (a), the experiments were carried out in triplicate. (c) The image of an agarose gel after fractionation by electrophoresis and SYBR Green I staining of deproteinized cohesive end ligation products from reactions containing different X4L4 constructs and different XLF mutants as indicated in the presence and the absence of 4% of PEG8000. The substrate was 2.7 kb XbaI-linearized pUC19 plasmid. The reaction time was 15 minutes. The protein concentrations were fixed at 2 μM. Statistics with unpaired two-tailed t-test, ***P < 0.001, n=3. (d) The quantification of the ligated DNA products of the reactions shown in (c), n=4 independent experiments. Statistics with unpaired two-tailed t-test. ****P < 0.0001, ***P < 0.001, ns = not significant. Data are represented as mean values ± SD.
Supplementary information
Supplementary Information
Supplementary Methods, Figs. 1–14 and Tables 1–4.
Supplementary Data 1
Comprehensive list of plasmid constructs used in this study.
Supplementary Data 2
Source data for Supplementary Figs. 1–14.
Source data
Source Data Fig. 1
Statistical source data for Fig. 1d.
Source Data Fig. 1
Unprocessed western blots for Fig. 1e.
Source Data Fig. 2
Statistical source data for Fig. 2d,f,g,j.
Source Data Fig. 2
Unprocessed western blots for Fig. 2k.
Source Data Fig. 3
Statistical source data for Fig. 3d.
Source Data Fig. 4
Statistical source data for Fig. 4d,g.
Source Data Fig. 5
Statistical source data for Fig. 5b,e.
Source Data Extended Data Fig. 1
Statistical source data for Extended Data Fig. 1, left.
Source Data Extended Data Fig. 1
Unprocessed western blots for Extended Data Fig. 1, right.
Source Data Extended Data Fig. 2
Statistical source data for Extended Data Fig. 2a–e.
Source Data Extended Data Fig. 3
Statistical source data for Extended Data Fig. 3b–d.
Source Data Extended Data Fig. 4
Statistical source data for Extended Data Fig. 4a–d.
Source Data Extended Data Fig. 5
Statistical source data for Extended Data Fig. 5c.
Source Data Extended Data Fig. 7
Statistical source data for Extended Data Fig. 7.
Source Data Extended Data Fig. 8
Statistical source data for Extended Data Fig. 8b.
Source Data Extended Data Fig. 9
Statistical source data for Extended Data Fig. 9b.
Source Data Extended Data Fig. 10
Statistical source data for Extended Data Fig. 10b,d.
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Vu, DD., Bonucci, A., Brenière, M. et al. Multivalent interactions of the disordered regions of XLF and XRCC4 foster robust cellular NHEJ and drive the formation of ligation-boosting condensates in vitro. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01339-x
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DOI: https://doi.org/10.1038/s41594-024-01339-x
