Multivalent interactions of the disordered regions of
XLF and XRCC4 foster robust cellular NHEJ and drive
the formation of ligation-boosting condensates in vitro

Duc-Duy Vu, Alessio Bonucci, Manon Brenière, Metztli Cisneros-Aguirre, Philippe Pelupessy, Ziqing Wang, Ludovic Carlier, Guillaume Bouvignies, Patricia Cortes, Aneel K Aggarwal, et al.

► To cite this version:

Duc-Duy Vu, Alessio Bonucci, Manon Brenière, Metztli Cisneros-Aguirre, Philippe Pelupessy, 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. Nature Structural and Molecular Biology, 2024, 31 (11), pp.1732-1744. 10.1038/s41594-024-01339-x. hal-05183999

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# Multivalent interactions of the disordered regions of XLF and XRCC4 foster robust cellular NHEJ and drive the formation of ligation-boosting condensates in vitro

Received: 27 June 2023

Accepted: 28 May 2024

Published online: 19 June 2024

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A list of authors and their affiliations appears at the end of the paper

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.

DNA double-strand breaks (DSBs) can be induced by external agents, such as ionizing radiation, or by internal processes, including errors in DNA transactions and the action of reactive oxygen species produced by oxidative metabolism¹. DSBs also form as intermediates during programmed genome rearrangements in developing lymphocytes¹. If undetected or misrepaired, DSBs threaten genomic integrity as they may lead to chromosomal rearrangements that can trigger tumor formation or cell death. Several pathways have evolved to mend DSBs,

among which the non-homologous end joining (NHEJ) pathway is primordial in mammalian cells¹⁻³.

NHEJ is a flexible process in terms of both structures and mechanisms¹⁻³. NHEJ is rapid and adaptable to repair any type of broken DNA end structures such as blunt or hairpin-ended DNA, as well as DNA ends with overhangs and DNA ends with complex damages. This is achieved by the assembly of an untemplated synaptic scaffold that brings and holds together the two free broken DNA ends, recruits end-processing

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e-mail: mauro.modesti@inserm.fr; fabien.ferrage@ens.psl.eu
<figure><img src="image_4.png" /><figcaption>**Fig. 1** | The CTRs of XLF and XRCC4 promote robust cellular NHEJ. **a**, Left, domain organization of XLF, XRCC4 and LIG4 proteins, where XLF and XRCC4 form homodimers. Right, cartoon representation of the NHEJ short-range synapsis model formed by the indicated proteins and DNA ends. The blue, red, and gray ovals on the CTRs of XLF and XRCC4 indicate the positively charged, negatively charged and hydrophobic regions, respectively (see Figs. 2i and 3d and Supplementary Fig. 1d for the details of region boundaries). **b**, The crystal structure of XLF (PDB 2R9A) and the cryo-EM structure of X4L4 (PDB 7LSY). The dashed lines represent the CTRs that are lacking in these structures but visible in the NMR spectra in Figs. 2a and 3a for XLF and XRCC4, respectively. **c**, The EJ7-GFP reporter (not to scale) for no-indel end joining between two Cas9-sgRNA DSBs that was integrated into XRCC4-KO XLF-KO HEK293 cells. **d**, GFP frequencies are normalized to parallel GFP transfections to account for transfection efficiency. **n** = 9 biologically independent transfections. Statistical significance was determined by an unpaired two-tailed *t*-test with Holm–Šidák correction. The fold effect is represented. \*\*\*P < 0.0001, \*\*P < 0.001 and \*P < 0.01; NS, not significant. The exact *P* values from top to bottom are <0.0001, 0.0532, 0.0532, 0.0002, <0.0001, <0.0001 and <0.0001, respectively. Data are represented as the mean values ± s.d. **e**, Immunoblots show the levels of XRCC4<sub>WT</sub>, XRCC4<sub>1-230</sub>, XLF<sub>WT</sub> and XLF mutants. The asterisk indicates background XLF or XRCC4.</figcaption></figure>

factors and ultimately promotes the ligation step¹⁻³. The dynamic nature and the involvement of numerous effectors of NHEJ render the investigation of this mechanism challenging, especially in determining how the core effectors assemble and coordinate to ensure effective DNA repair.

Many contributions from structural and single-molecule studies have yielded clear pictures of several essential steps in the NHEJ pathway. In the consensus model, the Ku70–Ku80 heterodimer (Ku) initially threads onto broken DNA ends and directly attracts the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), X4L4 (X-ray repair cross complementing 4 (XRCC4) in complex with DNA ligase 4 (LIG4)) and XLF (XRCC4-like factor, also known as NHEJ factor 1 or Cernunnos) to assemble into a synaptic complex that tethers and religates DNA ends¹⁻⁹. Single-particle cryo-electron microscopy (cryo-EM) studies of NHEJ supercomplexes containing Ku, XLF, XRCC4 and LIG4 with or without DNA-PKcs assembled on DNA have revealed several structural organizations of the NHEJ synaptic scaffold and suggested how transitions between different states may occur⁸,¹⁰⁻¹⁶. However, the putatively disordered C-terminal regions (CTRs) of both XLF and

XRCC4 are mostly unresolved, likely because of their heterogeneous conformations under cryogenic conditions.

In earlier studies, the CTRs of XLF and XRCC4 were considered dispensable for NHEJ activities but recent studies have indicated otherwise¹⁴,¹⁷,¹⁸, suggesting that their functional roles in NHEJ are complex and intricate. Substitutions in the CTR of XRCC4 or its deletion did not affect X-ray sensitivity or variable–diversity–joining (V(D)J) recombination in cells¹⁹,²⁰ yet were important for DNA interaction in vitro²¹. Likewise, deletion of the CTR of XLF appeared dispensable for its activity during DNA repair in cells²² and is, on the other hand, important for end joining in vitro²³. Furthermore, the CTRs of XRCC4 and XLF are targets for phosphorylation by DNA-PK and ataxia telangiectasia mutated protein but these phosphorylation sites were not required for DNA repair in cells²⁴,²⁵. Other phosphorylation sites by casein kinase 2 in the XRCC4<sub>CTR</sub> or by protein kinase B in the XLF<sub>CTR</sub> have been implicated in interactions with DNA end-processing factors²⁶ or cellular localization²⁷, respectively. More recently, finer understanding of the roles of the XRCC4 and XLF CTRs has emerged through the characterization of their involvement in a number of weak and transient interactions in
NHEJ complexes<sup>13,17,18</sup>. Thus, the CTRs of XLF and XRCC4 have regulatory roles during DNA repair controlling scaffolding and DNA interactions, recruitment of factors and cellular localization. However, the functional roles of the CTRs of XLF and XRCC4 during NHEJ remain elusive. Here we use cellular models to show that the CTRs of XRCC4 and XLF are crucial for robust NHEJ activity. We use nuclear magnetic resonance (NMR) to understand their functional role at residue resolution. We demonstrate that the CTRs of both XLF and XRCC4 are intrinsically disordered and form a network of weak multivalent interactions that drives the formation of condensates of XLF and X4L4 in vitro. XLF and X4L4 condensates recruit other NHEJ factors and strongly stimulate DNA end ligation, offering a yet unexplored perspective on the organization in space and time of the DSB repair mechanism by NHEJ.

## Results

### The CTRs of XLF and XRCC4 promote robust cellular NHEJ

XLF and XRCC4 share a similar structural organization, including a head domain, a coiled-coil domain (CC) and a CTR (Fig. 1a,b)<sup>23,28</sup>. To test the importance of the CTRs of XLF and XRCC4 for cellular NHEJ, the EJ7-GFP (green fluorescent protein) reporter was used to measure end joining between distal ends of two Cas9-mediated blunt chromosomal DSBs, without indel mutations from the edge of the DSB (Fig. 1c). The importance of blunt DSB end joining without indels is that such repair is not stabilized by an annealing intermediate and has been shown to be dependent on NHEJ<sup>29</sup>. No-indel end joining causes restoration of a GFP-positive cassette that is measured by flow cytometry and this repair event has previously been shown to require both XRCC4 and XLF<sup>29</sup>. To examine substitutions in both factors simultaneously, we used a previously described XRCC4-KO XLF-KO HEK293 cell line with an EJ7-GFP reporter integrated in the genome<sup>30</sup>, which was transfected with the plasmids for Cas9-sgRNAs to induce DSBs, along with expression vectors for XLF and XRCC4 and the truncated mutants XLF<sub>1-251</sub> and XRCC4<sub>1-230</sub>. For XLF, we also combined the truncation variant with L115A, which weakens the binding interface with XRCC4 (refs. 23,29,31,32). Of note, all XRCC4 and XLF expression constructs expressed untagged versions of the proteins. We found that combining the deletion of both CTRs with the XLF<sub>1-251</sub> L115A mutant and XRCC4<sub>1-230</sub> caused a marked (9.8-fold) decrease in no-indel end joining compared to the wild type (XRCC4<sub>WT</sub>) (Fig. 1d-e).

The XRCC4<sub>1-230</sub> mutant also showed a significant but modest decrease versus XRCC4<sub>WT</sub> when combined with XLF, XLF<sub>L115A</sub> or XLF<sub>1-251</sub> (1.4-fold, 2.4-fold and 2.2-fold, respectively; Fig. 1d). Similarly, the XLF<sub>1-251</sub> L115A mutant showed a modest decrease versus XLF<sub>WT</sub> when combined with XRCC4<sub>WT</sub> (1.9-fold). Expression levels of the XRCC4<sub>1-230</sub> mutant appeared to be lower than for XRCC4<sub>WT</sub> (Fig. 1e); however, both levels were higher than for endogenous XRCC4, with which the XLF<sub>1-251</sub> L115A mutant caused only a 1.3-fold decrease (Extended Data Fig. 1), which is much smaller than the 9.8-fold decrease when combined with XRCC4<sub>1-230</sub> (Fig. 1d). We conclude that the CTRs of XLF and XRCC4 have important contributions in the promotion of robust cellular NHEJ. We

present below our NMR investigation of the CTRs of XLF and XRCC4 at residue resolution to decipher their functional roles.

### Interactions of the disordered CTR of XLF

The CTR of XLF is predicted to be disordered (Supplementary Fig. 1c). To confirm the disordered nature of this region with NMR spectroscopy, isotopically labeled full-length XLF and its CTR in isolation (residues 229–299, XLF<sub>CTR</sub>) were produced (Supplementary Fig. 1a). Despite the large molecular weight of the XLF homodimer (66 kDa), the ¹H–¹⁵N heteronuclear single quantum coherence (HSQC) spectrum of isotopically labeled full-length XLF showed sharp and well-resolved peaks (Fig. 2a). Most peaks of the spectra of full-length XLF and XLF-CTR alone were superimposed. We used a series of triple-resonance NMR experiments to assign backbone resonances of all non-proline residues in the CTR, between residues 232 and 299 (Supplementary Fig. 2a,b). ¹H chemical shift dispersion, secondary structural propensity, ¹⁵N–{¹H} heteronuclear nuclear Overhauser effect (hNOE) and small-angle X-ray scattering (SAXS) experiments jointly indicated the lack of stable secondary structure elements in the XLF<sub>CTR</sub> either alone or in the context of the full-length protein construct (Supplementary Fig. 3a,b and Extended Data Fig. 2), defining it as an intrinsically disordered region (IDR).

### Interaction of XLF with Ku

The NMR spectra of the disordered CTR within the full XLF homodimer make it possible to characterize interactions of the CTR, which were previously explored either indirectly or with reductions to short peptides<sup>33</sup>. The interaction of XLF and Ku is well known<sup>33</sup>. The Ku-binding motif (KBM) of XLF has been defined as the last ten residues of XLF and structures of Ku bound to XLF<sub>KBM</sub> peptide have been solved by X-ray crystallography<sup>33,34</sup>. We titrated ¹⁵N XLF<sub>CTR</sub> with the Ku80 von Willebrand domain (Ku80<sub>VWA</sub>) and observed that XLF–Ku interactions mainly involved the residue K293 and the last five residues (<sup>295</sup>RGLFS<sup>299</sup>), with notable chemical shift perturbations (CSPs) and peaks in the slow exchange regime, while residues 283–292 were only slightly perturbed upon binding to Ku80<sub>VWA</sub> (Fig. 2c,d). This is in agreement with the structure of Ku with the XLF<sub>287-299</sub> peptide where only the residues <sup>295</sup>RGLFS<sup>299</sup> occupy the pocket of Ku80<sub>VWA</sub> to stabilize the XLF–Ku interaction<sup>8,33</sup>, whereas the perturbation of K293 may be attributed to changes in conformational propensity because of the proximity of the binding site, as this residue did not form any interaction with Ku in the crystal or cryo-EM structures<sup>8,33</sup>. This led us to define the core KBM of XLF as residues R295–S299.

### Interaction of XLF with DNA

The CTR of XLF was shown to have an important role in the DNA-binding capacity of the full-length protein<sup>21,25</sup>. Yet, the region of interaction was not clear<sup>35</sup>. We monitored the CSPs of XLF upon the addition of DNA to better characterize the region of interaction of XLF with DNA (Fig. 2e,f and Extended Data Fig. 3). The presence of stoichiometric amounts of double-stranded DNA induced notable CSPs in the ¹H–¹⁵N

**Fig. 2 | Multivalent interactions within the CTR of XLF.** a, Overlay of ¹H–¹⁵N HSQC spectra of full-length XLF (cyan) and XLF<sub>CTR</sub> (red). b, Insets from superimposed ¹H–¹⁵N HSQC spectra of 250 µM XLF<sub>CTR</sub> (red) after adding one (orange) or two (pink) equivalents of XLF<sub>1-224</sub> compared to the spectrum of full-length XLF (cyan). c, Overlay of ¹H–¹⁵N HSQC spectra of 50 µM XLF<sub>CTR</sub> alone (red) and in the presence of one half (orange) or one (green) equivalent of Ku80<sub>VWA</sub>. d, Peak intensity ratios from ¹H–¹⁵N HSQC spectra of ¹⁵N XLF<sub>CTR</sub> alone (I<sup>o</sup>) and after adding one half (orange) or one (green) equivalent of Ku80<sub>VWA</sub>. e, Overlay of ¹H–¹⁵N HSQC spectra of 150 µM XLF recorded in a titration with 30mer DNA. f, CSPs of XLF upon addition of four equivalents of 30mer DNA. g, The residues of BRCT1 that experienced notable peak intensity reduction (I/I<sup>o</sup> < 0.5) after adding XLF<sub>CTR</sub> are mapped as a color gradient (0, blue; 0.5, purple) on the BRCT1 structure (generated by AlphaFold Multimer). I/I<sup>o</sup>, normalized peak intensity of BRCT1 before and after adding two equivalents of XLF<sub>CTR</sub>. h, The model of

BRCT1–XLF<sub>CTR</sub> complex predicted by AlphaFold Multimer<sup>44</sup>; the affected residues are color-mapped as in *g*, *i*, Sequence alignment of the last 20 residues of XLF homologs. Hydrophobic residues and glycine, orange; positively charged residues, blue; strictly conserved residues, red. The residues of the CTR of XLF that interact with different partners are indicated. *j*, Cellular NHEJ activity assay. GFP frequencies are normalized to parallel GFP transfections to account for transfection efficiency. *n* = 9 biologically independent transfections. The values of XLF<sub>EV</sub>, XLF<sub>WT</sub> and XLF<sub>L115A</sub> with or without XRCC4<sub>WT</sub> or XRCC4<sub>1-230</sub> are the same as those in Fig. 1d. Statistical significance was determined by an unpaired two-tailed *t*-test with Holm–Sidak correction. The fold effect is represented. ***P* < 0.0001, **P* < 0.001 and *P* < 0.01. The exact *P* values from top to bottom are <0.00013, 0.00452, 0.05318, 0.00018, <0.0001, <0.0001 and <0.0001, respectively. *k*, Immunoblots show the levels of XRCC4<sub>WT</sub>, XRCC4<sub>1-230</sub>, XLF<sub>WT</sub> and XLF mutants. Data are represented as the mean values ± s.d.
HSQC spectrum of XLF. The largest perturbations were observed for residues R283, V289, K290, R291 and K293, whereas smaller perturbations were detected for the residues in the KBM, suggesting that this motif does not notably contribute to DNA binding. Consistently, the K293A substitution greatly reduced the affinity of XLF for DNA, while the K290A;R291A double substitution abolished the interaction. In

contrast, the L297A and F298A substitutions in the KBM did not notably affect the interaction (Extended Data Fig. 3e). Therefore, the positively charged region (residues 282–294) is the core of the DNA-binding motif (DBM) (Fig. 2i).

XLF binds to DNA in a length-dependent manner<sup>25,36</sup>. The region 290–296 of XLF shows sequence similarities with AT-hook motifs

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<caption>**Fig. 3** | Multivalent interactions within the CTR of XRCC4. a, Overlay of ¹H–¹⁵N HSQC spectra of XRCC4<sub>CTR</sub> (red) and X4L4 (cyan). b, CW-EPR spectra (X-band) recorded at room temperature for XRCC4 variants C218-MTSL (orange), C299-MTSL (cyan) and C337-MTSL (gray). The simulated spectra and the simulation parameters adopted for each spectrum are reported in Supplementary Fig. 13. c, Left, inset showing a part of the superimposed spectra of ¹⁵N XRCC4<sub>CTR</sub> upon adding one (green) or two (pink) equivalents of DBD<sub>LIG4</sub>. Right, inset showing the same region of the superimposed spectra of ¹⁵N full-length X4L4 (cyan) or X4-BRCTs (orange). The shift of the peak corresponding to residue F333 of XRCC4<sub>CTR</sub></caption>

that preferentially bind to the minor groove of A+T-rich sequences of double-stranded DNA³⁷,³⁸. To decipher the binding mode of XLF to DNA, XLF was titrated with double-stranded DNA of different lengths (15mer, 18mer, 21mer and 30mer) and sequence compositions (A+T-rich, G+C-rich and random sequences) (Extended Data Fig. 3). The apparent dissociation constants ($K_D$) were quantified by fitting the two-dimensional (2D) lineshapes and the positions of the affected peaks assuming a two-state one-to-one binding model³⁹. The apparent $K_D$ values of XLF for double-stranded DNA with 21mer and 30mer ranged from 4 to 16 µM and increased to 34 and 141 µM for 18mer and 15mer DNA, respectively. This is in agreement with the length-dependent affinity of XLF for DNA, as observed in previous studies²⁵,³⁶. XLF had comparable affinities for A+T-rich, G+C-rich and random sequences (Extended Data Fig. 3), despite the similarity of the DBM of XLF to AT-hook motifs³⁷. To identify the binding mode of the CTR of XLF to DNA, we carried out a competitive binding experiment involving XLF, A+T-rich DNA and Netropsin (Supplementary Fig. 4), a small molecule that binds specifically to the minor groove of A+T-rich DNA⁴⁰. Netropsin competed efficiently with XLF on A+T-rich DNA, suggesting that XLF and Netropsin share the same binding site. Thus, residues 282–295 in the CTR of XLF bind to DNA, most likely in the minor groove, in a length-dependent and sequence-non-specific manner.

## XLF–XLF interactions

Next, we identified the presence of homotypic interactions in full-length XLF that involve the CTR. The backbone ¹⁵N transverse relaxation rates ($R_2$) in the DBM and KBM of the full-length XLF exhibited greatly elevated values compared to the XLF<sub>CTR</sub> alone (Extended Data Fig. 4a), suggesting transient interactions with the folded domain. Accordingly, adding the folded domain of XLF (XLF<sub>1–224</sub>) to a solution of ¹⁵N XLF<sub>CTR</sub> increased the values of $R_2$ for the residues in the DBM and KBM similarly to those in full-length XLF. In addition, ¹H and ¹⁵N chemical shifts of residues L297 and F298 shifted back toward the values in full-length XLF (Fig. 2b). These results demonstrate that the DBM and KBM of

is highlighted with an arrow. d, Top, $R_2$ of backbone ¹⁵N nuclei for residues corresponding to the XRCC4<sub>CTR</sub> in the X4-BRCTs complex at concentrations 600 and 70 µM. Bottom, $R_2$ of backbone ¹⁵N nuclei for XRCC4 in the X4-BRCTs complex (300 µM) with KCl concentrations of 150 mM (green) and 500 mM (orange). $R_2$ values were measured at 298 K on an 800-MHz spectrometer. The four regions (FBM, SIM, DIM and NCT) are highlighted in gray. The three-residue rolling charge distribution is displayed on top of the graph. The error bar represents the s.e.m. with 1σ uncertainty estimated from the covariance matrix ($n$ = 1 independent experiment).

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XLF<sub>CTR</sub> interacted with the folded domain of XLF. Moreover, the KBM and DBM of XLF<sub>CTR</sub> interacted synergistically with the folded domain of XLF, as the interaction was abolished only when key residues in both the DBM (K290A; R291A) and the KBM (F298A) were substituted to alanine (Extended Data Fig. 4b). We also observed that a non-cleavable hexahistidine tag at the C terminus of XLF, as used in several previous studies¹⁸,²¹, severely altered the $R_2$ profile of XLF<sub>CTR</sub> (Supplementary Fig. 2c,d), possibly by artificially stabilizing these intramolecular interactions.

Where do the DBM and KBM bind onto the folded domain of XLF? The large molecular weight of the XLF dimer and the possible formation of non-specific oligomers at high concentrations²³ prevented us from directly observing the folded domain in the NMR spectra. As an alternative strategy, paramagnetic relaxation enhancement (PRE) experiments were used to identify the binding sites of DBM and KBM onto the folded domain of XLF (Supplementary Fig. 5). In these experiments, a paramagnetic probe (nitroxide) was attached to a cysteine side chain from the folded domain of a single-cysteine mutant of XLF. The strong dipolar interaction between ¹H nuclear spin and the nitroxide’s electron spin leads to decreased intensity in the ¹H–¹⁵N NMR spectrum for residues that are in the proximity, even transiently, of the nitroxide probe, typically within 15 Å (refs. 41,42). Peaks from the KBM motif lost intensity when the probe was attached to the head domain (C74), while peaks from the DBM weakened when the probe was attached to the CC domain (C188) (Supplementary Fig. 5). These PRE patterns corroborate our hypothesis that the DBM and the KBM of XLF<sub>CTR</sub> interact with the folded domain at distinct binding sites. In addition, we estimated the residue-specific affinity of the homotypic interaction from the *in trans* NMR titration experiment between the CTR alone and the folded domain of XLF. The interaction was weak, with dissociation constants in the millimolar range (Extended Data Fig. 4c,d). However, in the context of full-length XLF, the population of the bound form is estimated to be between 3% and 6%. Such interactions with low affinity and multivalency can become relevant when high protein concentrations are encountered (see below).
## Interaction of XLF with LIG4

We next investigated the role of the CTR of XLF in the interaction with the X4L4 complex. In pull-down assays, XLF interacted with LIG4 through the first BRCA1 C-terminal domain (BRCT1) and XRCC4-interacting domain (XID)¹⁴, but the binding site remains unknown. XLF forms oligomers with XRCC4 and X4-BRCTs through an XLF-XRCC4 head-to-head interaction²¹,³²,³⁵, as confirmed by ¹⁵N-edited diffusion measurements in solution (Supplementary Fig. 6f). Importantly, severe intensity losses were observed for the KBM and DBM of XLF upon the addition of X4-BRCTs (Supplementary Fig. 6a). Signals for the KBM and DBM were attenuated even when the XLF-XRCC4 head-to-head interaction was disrupted by the L115D substitution³² (Supplementary Fig. 6b,c) but not when the BRCT1 domain was removed (Supplementary Fig. 6d,e). Taken together, these data indicate a direct interaction between the KBM and DBM of XLF and the BRCT1 domain of LIG4.

To identify the XLF-binding region in the LIG4 BRCT1, the resonances of the LIG4 BRCT1 domain (LIG4 residues 654–759) were assigned (Extended Data Fig. 5a). Adding the XLF<sub>CTR</sub> to isotopically labeled BRCT1 led to a reduction in peak intensities and changes in chemical shifts (Fig. 2g and Extended Data Fig. 5b,c). Using AlphaFold Multimer⁴⁴, a model of the BRCT1-XLF<sub>CTR</sub> complex was computed (Extended Data Fig. 5d). Although the predicting confidence for XLF<sub>CTR</sub> is low, the predicted interface agrees with the perturbation in the NMR spectra for both proteins. In this model for the molecular basis of the XLF<sub>CTR</sub>-BRCT1 interactions (Fig. 2h), residue F298 of XLF forms hydrophobic interactions with residues H753 and F754, whereas K293 of XLF further stabilizes the complex through electrostatic interactions with residue D661 of LIG4. The results also explain why the CTR of XLF can indirectly stabilize the interaction between XLF and XRCC4 as observed previously¹⁷,⁴⁵. Altogether, we show that the disordered CTR of XLF is an interaction hub, which binds weakly to Ku, DNA, LIG4 and the folded domain of XLF in an intramolecular or intermolecular fashion. Considering that XLF is a homodimer, it can engage in interactions with two partners simultaneously, while the proximity of the DBM and KBM motifs likely prevents the interaction of one CTR with two binding partners simultaneously.

To probe the role of specific interactions of the CTR of XLF for cellular NHEJ activity, we designed a triple mutant of XLF, with the substitutions K290A and R291A in the DBM and F298A in the KBM (XLF<sub>AAA</sub>) that impaired the interaction of the CTR of XLF with DNA (Extended Data Fig. 3e), the folded domain of XLF (Extended Data Fig. 4b), the Ku80<sub>VWA</sub> domain (Fig. 2f)³³ and potentially the LIG4 BRCT1 domain (Fig. 2g,h). When tested for its ability to support NHEJ in cells with the EJ7-GFP reporter, the XLF<sub>AAA</sub> mutant behaved similarly to the XLF<sub>1-251</sub> truncation described above and notably caused a marked (8.9-fold) decrease in no-indel end joining when combined with L115A and XRCC4<sub>1-230</sub> (Fig. 2j,k). These results highlight the key role of these specific interactions of the CTR of XLF for robust NHEJ activity in cells.

## Interactions of the disordered CTR of XRCC4

The CTR of XRCC4 is longer than the CTR of XLF, encompassing residues 202–336, and is predicted to be disordered (Supplementary Fig. 1c). Low-resolution cryo-EM structure classification⁴⁶ suggested that the CTR of XRCC4 may fold back to the CC domain. To characterize the structural properties of the CTR of XRCC4, we used isotopically labeled full-length XRCC4 and a protein fragment corresponding to its CTR (XRCC4<sub>CTR</sub>). Two additional protein samples containing isotopically labeled X4L4 or the unlabeled X4-BRCTs were also studied (Supplementary Fig. 1b). The ¹H–¹⁵N HSQC spectrum of XRCC4 and XRCC4<sub>CTR</sub> displayed low proton chemical shift dispersion, a signature of disordered protein regions (Fig. 3a and Supplementary Fig. 2e–g), with no detectable signal from the folded domain. Inspection of the ¹H–¹⁵N HSQC spectra of all the constructs and complexes identified only small differences, indicating no major conformational changes in the CTR of XRCC4. All non-proline residues from 208 to 366 were assigned unambiguously except residues 273–278. (Supplementary

Fig. 2e). NMR relaxation and chemical-shift analyses together with SAXS experiments confirmed the intrinsically disordered nature of the CTR of XRCC4 across all the constructs (Supplementary Figs. 3c,d and 7). In addition, continuous-wave electron paramagnetic resonance (CW-EPR) spectra also showed that the binding to LIG4 BRCTs did not notably alter the dynamic properties of the CTR of XRCC4 (Fig. 3b).

On the basis of the sequence conservation and functional roles (Supplementary Fig. 1d), four subregions of XRCC4<sub>CTR</sub> were identified: the Forkhead-associated domain-binding Motif (FBM; residues 226–240)⁴⁷, the SUMO interaction motif (SIM; residues 256–261)³⁰, the DNA-PKcs interaction motif (DIM; residues 262–288)⁸,¹¹ and the negatively charged CTR (NCT; residues 316–336) (Fig. 3d and Supplementary Fig. 1d). When titrating the XRCC4<sub>CTR</sub> with 21mer double-stranded DNA, small changes in the chemical shift of residues within the DIM region were observed, suggesting that this region can bind transiently to DNA (Supplementary Fig. 8a,b). This is in line with our previous results showing that the CTR of XRCC4 does not contribute notably to the DNA-binding capacity of the full-length protein²⁰ but is important for the DNA-bridging property of XLF-XRCC4 complexes¹⁸.

The four conserved regions within the CTR of XRCC4 exhibited high backbone ¹⁵N R₂ values, indicating the likely presence of local interactions (Supplementary Fig. 7a,f). The R₂ values increased with increasing concentration (Fig. 3d, top), suggesting the presence of intermolecular interactions between XRCC4 dimers mediated by the CTR of XRCC4. Considering the uneven charge distribution in XRCC4<sub>CTR</sub>, we hypothesized that those interactions were driven by electrostatics. Indeed, increasing salt concentrations greatly decreased the R₂ value, indicating weakened interactions (Fig. 3d, bottom). To identify specific long-range interactions involving the CTRs of XRCC4 subregions, PRE experiments on X4-BRCTs complexes were carried out on full-length XRCC4 (Supplementary Fig. 9). Comparison of the experimental PRE results with those calculated from random unconstrained conformations revealed that the CTR of XRCC4 is not fully extended; instead, its four subregions interact with one another. To test whether these long-range interactions within the CTR of XRCC4 are intermonomer or intramonomer, we measured the PRE effects for the XRCC4<sub>CTR</sub> alone (Supplementary Fig. 10). Long-range interactions involving the four conserved regions were still observed with the XRCC4<sub>CTR</sub> fragment but to a lesser extent. To directly characterize the conformational space of XRCC4<sub>CTR</sub>, the ensemble of XRCC4 was generated using flexible-meccano and the ASTEROIDS algorithm⁴⁸ with backbone chemical shifts, intramolecular PRE values and SAXS data (Supplementary Fig. 11). The XRCC4<sub>CTR</sub> ensemble clearly showed the lack of a notably populated secondary structure, with only slightly elevated α-helical propensity in the NCT subregion and the presence of long-range contacts between the SIM-DIM subregions and the FBM-NCT subregions. In brief, these data demonstrated that the four subregions within the CTR of XRCC4 can form a network of electrostatic interactions within the same monomer, between two monomers within an XRCC4 dimer and between XRCC4 dimers.

Lastly, we identified that the CTR of XRCC4 interacts with the DNA-binding domain (DBD) of LIG4. The ¹H–¹⁵N HSQC signals from XRCC4 residues around F333 exhibited significant chemical shift and intensity perturbations between X4-BRCTs and X4L4 (Fig. 3c), suggesting an interaction of the CTR of XRCC4 with the catalytic domain or DBD of LIG4. Accordingly, titration of ¹⁵N XRCC4<sub>CTR</sub> with the unlabeled DBD of LIG4 reproduced the perturbations around the residue F333 as observed in X4L4 (Fig. 3c and Supplementary Fig. 8c,d), demonstrating the interaction between the CTR of XRCC4 and the DBD of LIG4.

## Liquid-liquid phase separation (LLPS) of XLF and the complex of XRCC4 and LIG4

Networks of weak multivalent interactions involving IDRs have been proposed to facilitate protein-protein and protein-nucleic acid interactions to increase local concentrations leading to the formation of
<figure><img src="image_16.png" /><figcaption>**Fig. 4** | X4L4 and XLF undergo LLPS in vitro. **a**, Colocalization of XLF with X4L4 and DNA within the droplets observed by the fluorescence microscopy with fluorescein-labeled XLF, Cy3-labeled X4L4 and Cy5-labeled DNA. **b**, Phase diagram of XLF with X4L4. The gray and green circles represent monophasic and biphasic solutions, respectively. The experiments were performed in duplicate and the conditions when numerous foci could be found were assigned as a biphasic solution. **c**, Top, droplet fusion event of X4L4 and XLF droplets. Bottom, the same images are plotted as an intensity gradient to highlight the contrast. **d**, FRAP experiments of X4L4-XLF droplets. The fluorescence intensity of the droplets was analyzed. The inset images illustrate a representative droplet at different time points. The red line indicates the FRAP curve fitted with exponential recovery (n = 9). The concentration of XLF and X4L4 was fixed</figcaption></figure>

at 20 µM with 1% fluorescein-labeled XLF. **e**, Ability of different XLF and X4L4 constructs to undergo LLPS. The concentrations of all constructs were fixed at 10 µM (see Extended Data Figs. 8 and 9 for a complete range of concentrations). **f**, 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, X4L4-XLF or T4 with an increasing amount of PEG8000. The substrate was a 2.7-kb XbaI-linearized pUC19 plasmid. The reaction time was 5 min. **g**, Quantification of the ligated DNA products of the reactions in **f**. The experiments were carried out in triplicate. **h**, The same reactions as in **f** with 1% Cy3-labeled X4L4 were visualized using fluorescence microscopy. Data are represented as the mean values ± s.d.

condensates, which can, in turn, undergo LLPS⁴⁹⁻⁵¹. The formation of protein or protein-RNA condensates has been observed at DNA repair sites inside cells⁵²⁻⁵⁴ but LLPS of NHEJ proteins has not yet been reported. We explored whether the many multivalent interactions involving the CTRs of XLF and XRCC4 could induce LLPS. In the presence of Ficoll 400 as a crowding agent, X4L4 phase-separated with XLF at concentrations as low as 500 nM and did not require DNA to undergo LLPS (Fig. 4a,b, Supplementary Fig. 12 and Extended Data Fig. 6a). However, DNA was able to partition into XLF and X4L4 droplets (Fig. 4a). The dense protein phase showed liquid properties as evidenced by the observation of droplet-like fusion events (Fig. 4c). To further characterize the dynamics of the dense phase, fluorescence recovery after photobleaching (FRAP) experiments were carried out. First, we

conducted a total FRAP experiment, where the entire droplets were bleached. The recovery happened within 1 min and the fitted mobile part was 45.53% ± 0.80% (Fig. 4d). Additionally, we performed partial FRAP experiments (Extended Data Fig. 7a,b), bleaching approximately half of the area of the droplets⁵⁵. The kymograph and the quantification of fluorescence intensity in the bleached and non-bleached halves revealed the dynamic reorganization of proteins within the condensates (Extended Data Fig. 7a,b), in agreement with the properties of LLPS⁵⁵. Additionally, the average dip intensity of the unbleached half was 0.24 ± 0.08, which is in the same range as other LLPS systems, such as heterochromatin protein 1α (ref. 55).

Next, we sought to determine the minimal components required for LLPS of XLF and X4L4. X4L4 alone can undergo phase transition
<figure><img src="image_17.png" /><figcaption>**Fig. 5** | Effect of other NHEJ components on the LLPS of XLF and X4L4.
a, Colocalization of X4L4 and Ku in the presence of XLF mutant (F298A, deficient in Ku binding) and WT XLF without or with the addition of DNA (blunt-ended Smal-linearized pUC19). Scale bars, 16 µm. b, Strip plot and box plot showing the quantification of the Ku enrichment in the puncta across multiple micrographs for each condition. Statistical significance was determined by a two-tailed *t*-test (**** *P* < 0.0001). The box outlines the interquartile range relative to the median, while the whiskers extend up to a maximum of 1.5 times the interquartile range beyond the box; any outliers are indicated as black dots. The experiments were carried out in duplicate. c, Colocalization of Art<sub>CTR</sub> in X4L4–XLF or XLF–X4-BRCTs droplets. Scale bars, 16 µm. d, Fluorescence images showing the effect</figcaption></figure>

of increasing amounts of Ku on the X4L4–XLF droplets. The concentrations of XLF and X4L4 were fixed at 2 µM. e, Quantification of the droplet area shown in d. Scale bar, 10 µm. Three micrographs from the duplicate experiments were analyzed together. Statistical significance was determined by a two-tailed *t*-test (**** *P* < 0.0001). f, The inset shows the peak of the F333 residues of XRCC4<sub>CTR</sub> extracted from the overlaid ¹H–¹⁵N HSQC spectra of XRCC4<sub>CTR</sub> alone or in the presence of DBD<sub>LIG4</sub> and Art<sub>CTR</sub>. g, Fluorescence microscopy images of 1% Cy3-labeled X4L4 in the absence or presence of XLF with increasing concentrations of Art<sub>CTR</sub>. The concentrations of X4L4 (complex) and XLF (monomer) were fixed at 5 µM. Scale bars, 16 µm. Data are represented as the mean values ± s.d.

but requires higher concentrations of X4L4 (around 1 µM) (Fig. 4b). Removing the catalytic domain of LIG4 from X4-BRCTs impaired its ability to form droplets possibly because of the interaction between the CTR of XRCC4 and the DBD of LIG4 (Supplementary Fig. 12). Adding XLF to this X4-BRCTs complex restored phase separation at an increased critical concentration of 2 µM (Supplementary Fig. 12 and Extended Data Fig. 8).

To determine the molecular interactions that govern LLPS, a series of XLF and XRCC4 mutants were investigated (Fig. 4e and Extended Data Figs. 8a,b and 9a,b). Truncating the CTR of XLF or introducing the triple substitution (K290A;R291A;F298A) impaired the formation of XLF and X4-BRCTs droplets, most likely because of the disruption of the interactions of the CTR of XLF with the folded domain of XLF and with BRCT1 of LIG4 (Fig. 2i). In contrast, substitutions in either the DBM (K293A or K290A;R291A) or the KBM (L297E or F298A) alone did not fully prevent phase separation (Fig. 4e and Extended Data Fig. 8a,b). The introduction of the L115D substitution in XLF effectively disrupted the interaction with XRCC4 (ref. 32),

resulting in the impairment of LLPS, which highlights the importance of the head-to-head domain interaction of XLF and XRCC4 in LLPS. On the other hand, the L115A substitution of XLF, which weakened the interaction between XLF and X4L4 (ref. 56), led to a slight reduction in the tendency to form LLPS, as evidenced by the observed droplet areas (Extended Data Fig. 8a,b).

In the case of XRCC4 and LIG4, the introduction of phosphomimetic substitutions (S315D;S320D;S327D;S328D)¹⁸ into the CTR of XRCC4 (X4<sub>p</sub>L4), along with the removal of the catalytic domain of LIG4 and the majority of residues in the CTR of XRCC4 (X4<sub>1–230</sub>L4), slightly diminished the tendency to form droplets (Extended Data Fig. 9a,b). Moreover, the removal of the BRCT1 or BRCT2 domains further increased the critical concentration (Extended Data Fig. 9a,b). Interestingly, XRCC4 alone, its complex with XID<sub>LIG4</sub> and the X4<sub>ΔCTR</sub>-BRCTs complex did not undergo LLPS with XLF but instead led to filamentous aggregates (Extended Data Fig. 9a,b). Notably, removing both the CTR and the BRCT-binding region of XRCC4 (XRCC4<sub>1–151</sub>) suppressed phase separation.
a

<figure><img src="image_18.png" /><figcaption>**Fig. 6 | Phase separation from the multivalent interactions of the CTR of XLF and XRCC4 and their role in NHEJ.** a, Summary of the multivalent interactions of XLF and XRCC4 CTRs characterized in this study (pink and blue wavy lines), as well as previously characterized interactions (straight gray lines). b, Condensates may form and enhance the assembly of NHEJ supercomplexes, DNA tethering, rates of enzymatic reactions and the recruitment of other NHEJ effectors through multivalent interactions of the IDRs, extending the proposed synaptic models</figcaption></figure>

b

<figure><img src="image_19.png" /></figure>

obtained from single-particle cryo-EM and single-molecule analysis⁴,⁸. This occurs when the local concentrations of XLF and X4L4 (in shadow) are above a critical concentration. WRN, Werner syndrome protein; Pol µ, DNA polymerase µ; Pol λ, DNA polymerase λ; TdT, terminal deoxynucleotidyl transferase; TDP1, tyrosyl-DNA phosphodiesterase 1; PAXX, paralog of XRCC4 and XLF; CYREN, cell-cycle regulator of NHEJ¹⁻³.

Many transient interactions of the CTRs of XLF and XRCC4 are mediated by electrostatic interactions (Figs. 2i and 3d), which are expected to be sensitive to ionic strength. Indeed, increasing the salt concentrations above physiological conditions ([KCl] > 300 mM) effectively dissolved the droplets (Extended Data Fig. 6b). On the other hand, weakening the hydrophobic interactions with hexane-1,6-diol only slightly altered the formation of condensates (Extended Data Fig. 6c). Taken together, these data demonstrate that the multivalent interaction network of the CTRs of XLF and XRCC4 is the driving force of the XLF-X4L4 LLPS in vitro.

The dramatic concentration increase in specific proteins in condensates may impact the kinetics and the molecular mechanism of NHEJ. Hence, we examined the effect of LLPS on NHEJ ligation activity using a simplified model condensate in vitro that comprised X4L4 alone or in a mixture with XLF (Fig. 4f–h). The concentration of the crowding agent (polyethylene glycol, PEG) was increased to induce LLPS, which induced a dramatic stimulation of DNA end ligation. At the critical concentrations (6% and 4% for X4L4 and X4L4-XLF, respectively), the droplets formed and accelerated intermolecular DNA ligation, most likely through increased local concentration (mass action), as seen in other coacervate droplets⁵⁷,⁵⁸. As a control, we added increasing amounts of PEG while monitoring DNA ligation by T4 DNA ligase. This led to a small linear increase in ligated DNA products, slightly favoring intramolecular ligation as a result of the excluded volume effect.

Next, a series of XLF and XRCC4 constructs were assessed for their role in stimulating DNA ligation within the condition of LLPS of XLF and X4L4 (Extended Data Fig. 10a–d). Truncating the CTR of XLF or introducing the triple substitution K290A; R291A; F298A impaired DNA ligation (Extended Data Fig. 10a,b). In contrast, truncating the CTR of XRCC4 modestly reduced the efficiency of ligation (Extended Data Fig. 10c,d). Expectedly, simultaneous truncation of the CTR in both XLF and XRCC4 resulted in a total impairment of DNA ligation. Notably, a phosphomimetic mutant of XRCC4 did not induce any changes in the

efficiency of DNA ligation (Extended Data Fig. 10c,d). Overall, these findings underscore the importance of the CTRs of XLF and XRCC4 in facilitating DNA ligation within the context of LLPS.

## Effects of relevant client proteins on LLPS of XLF and X4L4

Condensates can recruit and concentrate molecules with low valency, which are often referred to as clients⁵⁹. The clients are not the main driving force for LLPS but can partition into condensates and alter its properties⁶⁰. This aspect is highly relevant for NHEJ as the pathway involves the assembly and sequential action of a variety of proteins¹⁻³. To explore the effect of clients on X4L4-XLF condensates, we investigated interactions of XRCC4-XLF condensates with either Ku or the CTR of the protein Artemis (Art<sub>CTR</sub>) as models for client proteins. Ku is highly abundant in nuclei, threads on broken DNA ends and acts as a hub to recruit other NHEJ proteins⁶¹. Ku can form strong to weak interactions with DNA, XLF and the BRCT1 domain of LIG4 (refs. 5,33,62). Artemis is an endonuclease and exonuclease that forms a stable complex with DNA-PKcs and is recruited to DSB sites for DNA end processing¹,²,¹³. In Artemis, the Art<sub>CTR</sub>, which contains 307 residues, interacts with the DBD of LIG4 (refs. 63–66). Here we refer to Ku as a multivalent client, whereas Art<sub>CTR</sub> is a monovalent client. In colocalization assays, both Ku and Art<sub>CTR</sub> behaved as client proteins as they effectively partitioned into the X4L4-XLF droplets (Fig. 5a–c). The partition of Ku is dependent on DNA and XLF interactions as the Ku-binding-deficient variant of XLF (F298A) abolished the colocalization of Ku in X4L4-XLF droplets (Fig. 5a,b). On the other hand, Ku enrichment in X4L4-XLF droplets increased significantly in the presence of DNA (Fig. 5a,b). For Art<sub>CTR</sub>, the partition was driven by the interaction with DBD<sub>LIG4</sub>, as removing the DBD from LIG4 impaired the recruitment of Art<sub>CTR</sub> into the droplets (Fig. 5c). This suggests that X4L4-XLF condensates can selectively recruit relevant factors to DSB sites inside the droplets.

As expected for a multivalent client⁶⁰, the presence of Ku promoted more and larger X4L4-XLF droplets (Fig. 5d,e). In contrast, adding
excess amounts of the Art<sub>CTR</sub> dissolved the X4L4 droplets (Fig. 5g, top) because of competition with the interaction between the CTR of XRCC4 and the DBD of LIG4 at regions around residue F333 of XRCC4 (Figs. 3c and 5f), resulting in valency dilution<sup>60,67</sup>. Yet, the increased number and diversity of transient interactions in X4L4-XLF droplets prevented Art<sub>CTR</sub> from dissolving the droplets (Fig. 5g, bottom). These results demonstrate that LLPS of X4L4-XLF can be promoted or destabilized by multivalent or monovalent partners, respectively. These results are also compatible with a role of multivalent Ku in the stimulation of X4L4-XLF condensates at the sites of DSBs.

## Discussion

In the past few decades, investigations using diverse biophysical approaches have revealed key aspects of the NHEJ mechanism but the functional roles of IDRs have often been overlooked. Structural biology has often characterized short peptides from IDRs of NHEJ proteins bound to their folded partners with strong-to-intermediate affinities<sup>33,64,66,68</sup>, while few studies revealed the functions of IDRs in the context of full-length proteins<sup>17,18</sup>. Here we showed that NMR could reveal the dynamics and interactions of the IDRs of core NHEJ factors with residue resolution in the context of full-length proteins and their complexes. We defined several interactions of XLF and XRCC4 that are driven by their IDRs (Fig. 6a). We revealed the motifs in the CTR of XLF that interact with DNA, the folded domain of XLF and the BRCT1 domain of LIG4. In the case of XRCC4, we showed that its CTR can interact transiently with DNA and form a network of electrostatic intramolecular and intermolecular interactions. In addition, XRCC4<sub>CTR</sub> and Art<sub>CTR</sub> bind to the DBD of LIG4 in a competitive manner. Using cellular models, we also demonstrated that the CTRs of XLF and XRCC4 promote robust cellular NHEJ activity.

What could be the molecular mechanism underlying the important roles of these CTRs for NHEJ activity? The multiple weak interactions driven by the IDRs, as well as the head domains, can increase local concentrations at DNA repair sites through self-enrichment, which would, in turn, increase the rates of molecular collisions and favor ligation or act as hubs for the recruitment of accessory factors important for DNA end processing<sup>3,17,69</sup>. Low-affinity and promiscuous interactions of IDRs can promote rapid but reversible and exchangeable DNA binding, providing dynamic adaptability to the NHEJ process. As IDRs have the ability to move and screen space more freely, they can facilitate and accelerate the assembly of NHEJ supercomplexes akin to the fly-casting mechanism<sup>70</sup>, which may explain why IDRs accelerate the assembly of NHEJ synaptic complexes<sup>4,6,18,71</sup>.

Importantly, we provided evidence that this network of weak multivalent interactions readily and at relatively low concentrations leads to LLPS of X4L4 and XLF in vitro (Fig. 6b). We showed that these condensates can recruit other NHEJ factors and DNA and dramatically promote DNA ligation, likely governed by mass action, an effect previously observed in the context of local enrichment of proteins by networks of interactions of IDRs<sup>49,58</sup>. An interesting aspect of protein-DNA cocondensates is that they can have the ability to generate forces that can pull DNA together, which could be highly relevant to the NHEJ mechanism<sup>72</sup>.

Can NHEJ components form condensates in response to DSBs in cell nuclei? XLF and X4L4 with endogenous expression levels distribute homogeneously throughout the nucleus<sup>73,74</sup>. However, upon irradiation, XLF and X4L4 are in fast dynamic exchange between DSB sites and the surrounding as their half-times of recovery ($t_{1/2}$) in the FRAP experiments are shorter than 10 s (refs. 73,74). In addition, numerous XLF and X4L4 clusters can be observed in cells treated with radiomimetic drugs followed by cellular fixation and visualized by super-resolution microscopy<sup>33,75</sup>. Those clusters have been interpreted as XLF and XRCC4 filaments but could instead result from condensates. It is also known that 53BP1, a histone DNA damage-induced mark reader, shares functions with XLF and forms liquid compartments around DSB sites that facilitate NHEJ<sup>53,76,77</sup>. Interestingly, two other proteins,

SFPQ (proline-rich and glutamine-rich splicing factor) and NONO (non-POU domain-containing octamer-binding protein), known to form paraspeckles in the nucleoplasm<sup>78</sup>, functionally overlap with XLF in DSB repair by NHEJ<sup>79</sup>. Collectively, these results point toward a plausible intervention of condensates in NHEJ at the DSB sites in cells, a hypothesis that requires further investigation.

We dissected the role of individual interactions and found different critical concentrations for the phase separation of different constructs and mutants (Fig. 4e and Extended Data Figs. 8 and 9), suggesting that LLPS of NHEJ could be triggered and/or dissolved by altering the number of multivalent interactions either through post-translational modifications, such as SUMOylation<sup>30,80</sup>, or recruitment of auxiliary factors, such as aprataxin (APTX)-like and bifunctional polynucleotide phosphatase-kinase (PNKP)-like factor (APLF) and long non-coding RNAs (lncRNAs)<sup>54,81,82</sup>, as seen in other biological systems<sup>83</sup>. Indeed, emerging evidence suggests that NIHCOLE (ncRNA involved in NHEJ oncogenic ligation efficiency) or LINPI (lncRNA In NHEJ pathway 1) lncRNAs stimulate NHEJ through condensate formation<sup>24,82</sup>, yet the driving forces at the molecular level are unknown. Complexes with high stoichiometries, such as DNA-PKcs trimers, which have been observed recently<sup>84</sup>, could contribute to LLPS. Moreover, the micron-sized XLF and XRCC4 condensates observed in vitro here may directly link to submicron-sized complexes or oligomers in cells, as suggested by other studies<sup>85-87</sup>. Furthermore, we provided atomic information about the multivalent interactions of the CTRs of XLF and XRCC4 that will allow the generation of various separation-of-function mutations to test LLPS of NHEJ components directly in cells. Such experiments represent a great challenge because of the high functional redundancy of several NHEJ effectors<sup>76,88-91</sup>, the control of DSB induction<sup>92</sup> and the interplay with other DSB repair pathways such as alternative NHEJ or homology-driven pathways<sup>93</sup>.

In summary, we revealed a multivalent interaction network between NHEJ proteins and nucleic acids mediated by the CTRs of XLF and XRCC4 that are important for robust cellular NHEJ activity and for the formation of LLPS in vitro. We propose that the local enrichment of NHEJ proteins is a key feature of the mechanism of higher-order synaptic and catalytic assembly for DNA end processing and ligation. Moreover, these hubs or condensates could serve as a flexible platform for the timely recruitment of NHEJ factors and stimulation of DNA ligation while protecting the DNA ends from undesired enzymes. The minimal components and molecular interactions underlying NHEJ condensates identified here will serve as a foundation for further in vitro and cellular studies. Lastly, the emerging understanding of the role of NHEJ disordered protein regions offers an additional dimension in the way that NHEJ can be controlled in space and time, paving the way for potential biomedical applications.

## Online content

Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41594-024-01339-x.

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© The Author(s), under exclusive licence to Springer Nature America, Inc. 2024

Duc-Duy Vu<sup>1</sup>, Alessio Bonucci<sup>2,9</sup>, Manon Brenière<sup>3,9</sup>, Metztli Cisneros-Aguirre<sup>4,9</sup>, Philippe Pelupessy<sup>1</sup>, Ziqing Wang<sup>1</sup>, Ludovic Carlier<sup>1</sup>, Guillaume Bouvignies<sup>1</sup>, Patricia Cortes<sup>5</sup>, Aneel K. Aggarwal<sup>6</sup>, Martin Blackledge<sup>7</sup>, Zoher Gueroui<sup>8</sup>, Valérie Belle<sup>2</sup>, Jeremy M. Stark<sup>4</sup>, Mauro Modesti<sup>3</sup> & Fabien Ferrage<sup>1</sup>

<footnote n="1">¹Département de Chimie, LBM, CNRS UMR 7203, École Normale Supérieure, PSL University, Sorbonne University, Paris, France. ²Aix Marseille Univ, CNRS UMR 7281, BIP Bioénergétique et Ingénierie des Protéines, IMM, Marseille, France. ³Cancer Research Center of Marseille, Department of Genome Integrity, CNRS UMR7258, Inserm U1068, Institut Paoli-Calmettes, Aix-Marseille University, Marseille, France. ⁴Department of Cancer Genetics and Epigenetics, Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, CA, USA. ⁵Department of Molecular, Cellular and Biomedical Sciences, CUNY School of Medicine at City College of New York, New York, NY, USA. ⁶Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA. ⁷Institut de Biologie Structurale (IBS), Grenoble Alpes University, CNRS, CEA, Grenoble, France. ⁸PASTEUR, Département de Chimie, École Normale Supérieure, PSL University, Sorbonne University, CNRS, Paris, France. ⁹These authors contributed equally: Alessio Bonucci, Manon Brenière, Metztli Cisneros-Aguirre. e-mail: mauro.modesti@inserm.fr; fabien.ferrage@ens.psl.eu</footnote>
# Methods

## Plasmid constructs

The WT XLF open reading frame (ORF) was coupled with a 6xHis-tag and tobacco etch virus (TEV)-cutting motif at the N terminus and subcloned into pET16b. XLF mutants were generated by site-directed mutagenesis from the WT XLF. The XLF<sub>CTR</sub> (229–299) coding sequence was coupled with a SUMO tag and TEV-cutting motif at the N terminus and subcloned into pET-22b(+). The XRCC4 ORF was coupled with a 6xHis-tag and TEV-cutting motif at the N terminus and subcloned into pET28b(+). The XRCC4<sub>CTR</sub> (200–336) coding sequence was coupled with a maltose-binding protein (MBP) tag and TEV-cutting motif at the N terminus and subcloned into pMAL-c5x. The vWA domain of Ku80 (residues 1–245) coding sequence was coupled with an MBP tag, 8xHis-tag and TEV-cutting motif at the N terminus and subcloned into pMAL-c5x. The XLF<sub>CTR</sub>, XRCC4<sub>CTR</sub>, MBP-BRCTS cysteine-free and MBP-BRCT1<sub>654–759</sub> coding sequences were synthesized and sequenced by the Gene Universal Company. The XRCC4 quadruple phosphomimetic substitutions (S315D; S320D; S327D; S328D) were generated by site-directed mutagenesis from the WT XRCC4. Supplementary Table 4 and Supplementary Data provide a comprehensive list of the plasmid constructs used in this study.

## Protein expression and purification

**XLF and XLF mutants.** The plasmids containing the XLF and XLF mutants were transformed into *Escherichia coli* Novagen BL21 Rosetta(DE3) bacteria. The bacteria were grown at 37 °C in Luria-Bertani medium containing 75 µg ml⁻¹ ampicillin and 25 µg ml⁻¹ chloramphenicol until reaching an optical density at 600 nm between 0.6 and 0.8 and were subsequently induced with 500 µM IPTG and incubated overnight at 18 °C. The pellets were collected by centrifugation at 12,000g at 4 °C for 15 min and sonicated in buffer B1 (25 mM Tris-HCl pH 7.5, 1,000 mM NaCl, 2 mM DTT, 1 mM EDTA, 10 mM imidazole and 1 mM PMSF supplemented with complete EDTA-free Protease Inhibitor Cocktail). The supernatant was obtained by centrifuging at 35,000g for 5 min at 4 °C and loaded into a prepacked HisTrap excel 5-ml column pre-equilibrated with buffer A1 (25 mM Tris-HCl pH 7.5, 50 mM NaCl, 2 mM DTT, 1 mM EDTA and 1 mM PMSF) at the rate of 1 ml min⁻¹ and was washed extensively with buffer B1 until obtaining a stable ultraviolet (UV) signal. In the next step, the column was equilibrated with A1 buffer until the conductivity was less than 10 mS cm⁻¹. Then, the HiTrap 5-ml heparin HP column was attached below the Ni-column. The protein was eluted from the Ni-column to the heparin column with 25 ml of buffer A2 (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 2 mM DTT, 1 mM EDTA, 300 mM imidazole and 1 mM PMSF). Then, the Ni-column was detached and the inlet pump was changed to A1. The protein was eluted from the heparin column with a buffer gradient (0–60% B1 in 40 min). The partially truncated variant of XLF corresponded to the first peak and the second peak was the intact XLF. The His-tag was cut by adding 5% TEV (w/w) into the XLF sample while dialyzing in buffer A1 overnight. The following day, the protein was passed through a Ni-column to remove uncut XLF and TEV. After that, the flowthrough fractions that contained XLF were concentrated and passed through a size-exclusion chromatography (SEC) 26/60 S200 column pre-equilibrated with NMR buffer (20 mM Bis-Tris pH 6.5, 150 mM KCl, 1 mM DTT and 1 mM EDTA). The fractions corresponding to the peaks were identified by a 10% precast polyacrylamide gel, pooled together, concentrated using a concentrator (mol. wt. cutoff, 30 kDa), flash-frozen in liquid nitrogen and stored at –80 °C. The <sup>15</sup>N and <sup>15</sup>N–<sup>13</sup>C uniformly labeled samples were prepared using the Marley method⁹⁴ with <sup>15</sup>NH₄Cl and [¹³C]glucose as the sole nutrient sources of nitrogen and carbon, respectively. XLF<sub>CTR</sub> was expressed and purified using the same protocol as XLF with the exception that a 16/60 S75 column was used in the SEC.

**XRCC4<sub>CTR</sub>.** The plasmid was transformed into *E. coli* Novagen BL21 pLysS(DE3) bacteria. The non-labeled and the <sup>15</sup>N and <sup>15</sup>N–<sup>13</sup>C uniformly

labeled XRCC4<sub>CTR</sub> proteins were expressed using the same protocol as XLF. The XRCC4<sub>CTR</sub> supernatant was loaded into a prepacked MBPTrap HP 5-ml column pre-equilibrated with buffer A1 at the rate of 1 ml min⁻¹ and was washed extensively with buffer B1 until obtaining a stable UV signal. The protein was eluted from the column with buffer containing 70% A2-MBP (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 2 mM DTT, 1 mM EDTA and 30 mM maltose) and 30% B1. The MBP tag was cut by adding 5% TEV (w/w) into the protein sample while dialyzing in buffer A1 overnight. The following day, the protein was passed through a HiTrap heparin column to remove MBP and TEV. The fractions corresponding to the XRCC4<sub>CTR</sub> were concentrated and passed through an SEC 16/60 S75 column pre-equilibrated with NMR buffer. The Supplementary Methods describe the expression and purification of the other proteins.

## Microscopy imaging of LLPS

Unless indicated otherwise, all LLPS imaging was carried out with buffer consisting of 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 1 mM EDTA and 12.5% Ficoll 400. The components were mixed as indicated by the concentration, transferred into a covered glass-bottom 384-well black plate (Dutscher) and then imaged immediately at room temperature using fluorescence microscopy performed on an inverted Zeiss Z1 microscope equipped with a ×60 oil-immersion objective (pixel size, 0.267 µm). For the colocalization and FRAP experiments, a Zeiss LSM 710 META laser scanning confocal microscope equipped with a ×63 oil-immersion objective was operated at 20 °C (pixel size, 0.204 µm). LSM Zen 2012 software was used to control and set up experiments. To prevent evaporation and droplet drifting, imaging chambers attached on the cover slide (Bio-Labs secureSeal) were used to contain the samples. Images were analyzed using Fiji (version 1.53)⁹⁵. The FRAP experiments were performed at 20 °C and the photobleaching of X4L4–XLF<sub>fluorescein</sub> droplets was achieved using a circular region of interest that covered the whole droplet and a 488-nm laser. Fluorescence recovery was monitored by acquiring images (512 × 512 pixels) every second at a scanning speed of 0.48 µs per pixel for up to 4 min. The data were analyzed using ImageFRAP (https://imagej.net/Analyze_FRAP aeroplane with a Jython script). The partial FRAP experiments were analyzed using a script obtained from the literature⁵⁵. To quantify the droplet areas, at least two and up to ten micrographs from at least two independent experiments were analyzed. The background was subtracted using the default parameters and the threshold was automatically set using the triangle method. Then, the images were converted into binary. The particle analysis function in Fiji was used to count and segment the droplet areas with the following parameters: size, 5–∞ pixels; circularity, 0.01–1.00.

## Biochemical assay of DNA end ligation under LLPS conditions

Reaction mixtures (10 µl) contained 40 ng of XbaI-linearized (cohesive ends) pUC19 plasmid, 10 mM MgCl₂, 1 mM adenosine triphosphate (ATP), 150 mM NaCl, 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM DTT, 2 µM X4L4 or 8 U per µl T4 DNA ligase (New England BioLabs) and an increasing amount of PEG8000 (w/v) as indicated. Then, 2 µM XLF was added when indicated. The reactions were incubated at room temperature for 5 min. Then, the mixtures were deproteinized by adding pronase E (4 µg µl⁻¹) and 1× purple gel loading dye (New England BioLabs) and incubated at 55 °C for 30 min. The reaction products were analyzed by 0.8% agarose gel electrophoresis using TBE buffer and stained with SYBR Green I. Gel images were visualized and quantified by Gel Doc XR+ and Image Lab. Each reaction was carried out in triplicate. The reactions were also monitored by a fluorescence microscope with a ×60 magnification objective using similar conditions but without adding ATP and MgCl₂.

## Cellular GFP reporter NHEJ assay

The HEK293 parental, XLF-KO and XRCC4-KO XLF-KO cell lines containing the EJ7-GFP reporter were previously described, along with the Cas9–sgRNA plasmids (7a and 7b) used to induce the DSBs in the
reporter<sup>30,96</sup>. The XRCC4 and XLF expression constructs were inserted in the pCAGGS-BSKX expression vector, which was also the empty vector (EV) control<sup>97</sup>. The reporter cell lines were seeded at 0.5 × 10<sup>5</sup> onto a 24-well plate. The cells were transfected on the following day with 200 ng of the 7a and 7b sgRNA-Cas9 plasmids, along with 50 ng of the XRCC4 expression vector or EV control and 50 ng of the XLF expression vector or EV control. Each well contained 1.8 µl of Lipofectamine 2000 (Thermofisher) in 0.5 ml of antibiotic-free medium. Cells were incubated for 4 h with the transfection mixture and then washed and fed with complete medium. Parallel transfections using a GFP expression vector and EV control (200 ng each) in place of the Cas9-sgRNA plasmids were used to normalize for transfection efficiency. The cells were fixed with 3% formalin 3 days after the transfection and analyzed with ACEA Quanteon (Agilent NovoExpresss Version 1.5.0), as described<sup>29,98</sup>. For immunoblotting, HEK293 cells were lysed with erythrocyte lysing buffer (250 mM NaCl, 5 mM EDTA, 50 mM HEPES, 0.1% (v/v) IGEPAL and Roche protease inhibitor) and sonicated with the Qsonica Q800R. Transfections were scaled to a six-well plate with HEK293 cells, using the EV to replace the sgRNA-Cas9 plasmids. Blots were probed with antibodies for XRCC4 (CBMAB-X0197-YC, 1:2,000), XLF (Antibodies ABIN2857060, 1:2,000) and MLH1 (Abcam ab92312, 1:1,000). Agilent NovoExpress version 1.5.0 for Quanteon was used to capture and analyze the flow cytometry data (Supplementary Fig. 14). Statistical analysis was performed with Prism version 8.3.0.

NMR

**XLF and XLF<sub>CTR</sub>.** Unless specified otherwise, the NMR spectra were acquired at 298 K on a Bruker 18.8-T (800 MHz proton frequency) spectrometer equipped with a room-temperature triple-resonance (TXI) probe. The NMR data were processed with NMRPipe<sup>99</sup> and then analyzed and visualized using NMRFAM-Sparky<sup>100</sup>, Matplotlib 3.8, Seaborn 0.13 and Python 3. The buffer used for the samples was 20 mM Bis-Tris pH 6.5, 150 mM KCl, 1 mM EDTA, 1 mM DTT and 5% D<sub>2</sub>O. To assign the CTR of XLF, a series of three-dimensional (3D) band-selective excitation short-transient (BEST) HNCA, HNCACB and HNCO experiments<sup>101-103</sup> was recorded on a <sup>13</sup>C, <sup>15</sup>N uniformly labeled sample of XLF with a concentration of 250 µM. The resonance assignments of XLF<sub>CTR</sub> were transferred from the full-length XLF. HNCACB, HNcoCACB and HNcaCO experiments were recorded on an XLF<sub>CTR</sub> sample to confirm the assignment transfer from full-length XLF.

**XRCC4<sub>CTR</sub>, XRCC4 and X4-BRCTs.** Because the ¹H–¹⁵N HSQC spectra of XRCC4<sub>CTR</sub>, XRCC4 and X4-BRCTs were very similar, the resonance assignment was performed on XRCC4<sub>CTR</sub> and was subsequently transferred unambiguously to the other constructs. A series of 3D BEST HNCA, HNcoCA, HNCACB and HNcoCACB and a series of 3D BEST-TROSY (transverse relaxation optimized spectroscopy) HNcoCA, HNcaNh and HNcacoNh experiments were acquired for this purpose<sup>101–103</sup>. Moreover, a series of 3D BEST HNCACB and HNcoCACB experiments was acquired on the <sup>13</sup>C, <sup>15</sup>N uniformly labeled X4-BRCTs sample to confirm the transferred assignment from XRCC4<sub>CTR</sub> and to determine the chemical shift of the CA and CB resonances. The BRCT1 domain was assigned using a similar series of 3D experiments to that used for XRCC4<sub>CTR</sub>.

**NMR relaxation measurement.** Unless specified otherwise, the NMR relaxation experiments were acquired on an 800-MHz spectrometer at 298 K using uniformly ¹⁵N-labeled samples. The ¹⁵N longitudinal relaxation rates ($R_1$) were acquired by sampling the decay of the magnetization at six different delays (for XLF) or ten delays (for XRCC4 constructs) between 0 and 1.8 s. All the ¹⁵N $R_2$ values were measured using the $R_2^{CPMG}$ (Carr–Purcel–Meiboom–Gill) experiments<sup>104</sup>, except for XRCC4<sub>CTR</sub> and XRCC4 in complex with the BRCTs domain, in which both $R_2^{CPMG}$ and $R_2^{echo}$ experiments were used and explicitly indicated. The $R_2^{echo}$ experiment used a single echo while decoupling the protons during the entire relaxation period, whereas a CPMG train of ¹⁵N

$\pi$-pulses interleaved with ¹H $\pi$-pulses was used in the $R_2^{CPMG}$ experiment<sup>104,105</sup>. Six delays between 4 and 100 ms were used to determine $R_2^{CPMG}$, while seven delays between 9 and 288 ms were used for the $R_2^{echo}$ experiments. ¹H–¹⁵N hNOE data were measured by detecting the ¹⁵N steady-state polarization while saturating the protons with a train of $\pi$-pulses and comparing the peak intensities to those of an experiment where the magnetization of ¹⁵N returned to equilibrium<sup>106</sup>.

**NMR titration experiments.** For the DNA–protein titration experiments, DNA (purchased from Eurogentec) was added to the isotopically labeled proteins after exchanging them into the NMR buffer. For protein–protein titration experiments, the unlabeled proteins were added to the solution of the isotopically labeled proteins. CSP values were calculated for all residues by subtracting the shifts of the proteins in the mixtures with DNA or partner proteins from the shifts of the protein alone, using the following equation: $CSP = \sqrt{((\Delta H^{\mathrm{N}})^2 + (0.1\Delta H^{\mathrm{N}})^2)/2}$. The apparent affinity of XLF ($K_D^{\mathrm{APP}}$) for DNA was determined by fitting the 2D lineshape of affected peaks using TITAN software<sup>107</sup>. The simple binding model was used and the four most affected and isolated peaks (V289, K290, K293 and R295) were used to fit the data. The ¹⁵N relaxation of the free form of XLF was fixed as the values measured from the $R_2$ experiment of XLF alone. A total of 100 bootstrap steps were used to estimate the standard deviation. The fitted parameters are included in the Source Data file.

The Supplementary Methods describe the PRE experiments and the estimation of the apparent *in trans* $K_D$ values for XLF homotypic interactions from ¹⁵N relaxation rates.

**Statistics and reproducibility**

All the fluorescence microscopy experiments were performed at least two times with similar results. Statistical significance was determined by an unpaired two-tailed *t*-test. The sample sizes were selected for all experiments on the basis of experience from our laboratory and other studies with similar assays to ensure reproducibility and statistical significance. For NMR experiments, each spectrum was recorded as a single independent experiment. The s.d. presented for all NMR-derived data was a direct propagation of the noise in each individual spectrum.

**Reporting summary**

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

**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.

**References**

94. Marley, J., Lu, M. & Bracken, C. A method for efficient isotopic labeling of recombinant proteins. *J. Biomol. NMR* **20**, 71–75 (2001).

95. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. *Nat. Methods* **9**, 676–682 (2012).

96. Bhargava, R., Lopezcolorado, F. W., Jillianne Tsai, L. & Stark, J. M. The canonical non-homologous end joining factor XLF promotes chromosomal deletion rearrangements in human cells. *J. Biol. Chem.* **295**, 125–137 (2020).
97. Stark, J. M., Pierce, A. J., Oh, J., Pastink, A. & Jasin, M. Genetic steps of mammalian homologous repair with distinct mutagenic consequences. *Mol. Cell. Biol.* **24**, 9305–9316 (2004).

98. Gunn, A. & Stark, J. M. I-Scel-based assays to examine distinct repair outcomes of mammalian chromosomal double strand breaks. *Methods Mol. Biol.* **920**, 379–391 (2012).

99. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. *J. Biomol. NMR* **6**, 277–293 (1995).

100. Lee, W., Tonelli, M. & Markley, J. L. NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy. *Bioinformatics* **31**, 1325–1327 (2015).

101. Favier, A. & Brutscher, B. NMRlib: user-friendly pulse sequence tools for Bruker NMR spectrometers. *J. Biomol. NMR* **73**, 199–211 (2019).

102. Solyom, Z. et al. BEST-TROSY experiments for time-efficient sequential resonance assignment of large disordered proteins. *J. Biomol. NMR* **55**, 311–321 (2013).

103. Schanda, P., Van Melckebeke, H. & Brutscher, B. Speeding up three-dimensional protein NMR experiments to a few minutes. *J. Am. Chem. Soc.* **128**, 9042–9043 (2006).

104. Kay, L. E., Nicholson, L. K., Delaglio, F., Bax, A. & Torchia, D. A. Pulse sequences for removal of the effects of cross correlation between dipolar and chemical-shift anisotropy relaxation mechanisms on the measurement of heteronuclear *T*₁ and *T*₂ values in proteins. *J. Magn. Reson.* **97**, 359–375 (1992).

105. Ferrage, F. Protein dynamics by ¹⁵N nuclear magnetic relaxation. *Methods Mol. Biol.* **831**, 141–163 (2012).

106. Ferrage, F., Reichel, A., Battacharya, S., Cowburn, D. & Ghose, R. On the measurement of ¹⁵N–{¹H} nuclear Overhauser effects. 2. Effects of the saturation scheme and water signal suppression. *J. Magn. Reson.* **207**, 294–303 (2010).

107. Waudby, C. A. & Christodoulou, J. NMR lineshape analysis of intrinsically disordered protein interactions. *Methods Mol. Biol.* **2141**, 477–504 (2020).

## 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.

## Author contributions

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.

## Competing interests

The authors declare no competing interests.

## Additional information

Extended data is available for this paper at
https://doi.org/10.1038/s41594-024-01339-x.

**Supplementary information** The online version contains supplementary material available at https://doi.org/10.1038/s41594-024-01339-x.

**Correspondence and requests for materials** should be addressed to Mauro Modesti or Fabien Ferrage.

**Peer review information** *Nature Structural & Molecular Biology* thanks Amanda Chaplin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Dimitris Typas, in collaboration with the *Nature Structural & Molecular Biology* team.

**Reprints and permissions information** is available at
www.nature.com/reprints.
<figure><img src="image_20.png" /></figure>

**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

<figure><img src="image_21.png" /></figure>

*XLF AAA = XLF K290/R291/F298A mutation

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.
a

<figure><img src="image_22.png" /></figure>

b

<figure><img src="image_23.png" /></figure>

c

<figure><img src="image_24.png" /></figure>

d

<figure><img src="image_25.png" /></figure>

e

<figure><img src="image_26.png" /></figure>

**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.
<figure><img src="image_27.png" /></figure>

**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 ($K_D^{app}$ ± 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.
<figure><img src="image_28.png" /></figure>

**Extended Data Fig. 4** | XLF<sub>CTR</sub> interacts with the folded domain. Only the peaks belonging to the residues in the CTR are visible and analyzed.

(a) Transverse relaxation rate ($R_2$) of XLF<sub>CTR</sub> (green) after adding 1 (orange) or 2 (pink) equivalents of XLF<sub>1-224</sub> compared with full-length XLF (red). (b) $R_2$ 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)<sup>15</sup>N $R_2$ values of the most affected residues in XLF<sub>CTR</sub> plotted against the concentration of XLF<sub>1-224</sub> at a constant

XLF<sub>CTR</sub> concentration of 250 µM. (d) Estimation of the apparent residue-specific dissociation constants from the in trans titration. The $K_D$ values (bar plot, black) are extracted from the slopes of the $R_2$ vs. concentration in XLF<sub>1-224</sub> plotted in (c). The in trans residue-specific apparent dissociation constants $K_D$ and the $R_2$ 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.
a

<figure><img src="image_29.png" /></figure>

c

<figure><img src="image_30.png" /></figure>

b

<figure><img src="image_31.png" /></figure>

d

<figure><img src="image_32.png" /></figure>

**Extended Data Fig. 5** | Identifying the region of BRCT1 that interact with XLF<sub>CTR</sub>. (a) $^{1}$H-$^{15}$N HSQC spectra of BRCT1 (residues 654-759) with resonance assignment. (b) Overlay $^{1}$H-$^{15}$N HSQC spectra of BRCT1 in the absence (orange) and in the presence (cyan) of two equivalents of XLF<sub>CTR</sub>. (c) top: normalized peak

intensity ratios extracted from $^{1}$H-$^{15}$N HSQC spectra of $^{15}$N BRCT spectra before (I) and after adding 1 (green) or 2 (cyan) equivalents of XLF<sub>CTR</sub> (I), bottom: chemical shift perturbation (CSP) of BRCT1 after adding 2 equivalents of XLF<sub>CTR</sub>. (d) BRCT1-XLF<sub>CTR</sub> complex predicted using AlphaFold-Multimer.
<figure><img src="image_33.png" /></figure>

**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.
a

<figure><img src="image_34.png" /></figure>

b

<figure><img src="image_35.png" /></figure>

**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.
<figure><img src="image_36.png" /></figure>

b

<figure><img src="image_37.png" /></figure>

**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-BRTCS, 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.
<figure><img src="image_38.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_39.png" /></figure>

X4L4 constructs

**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 X4<sub>1-203</sub>-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.
<figure><img src="image_40.png" /></figure>

**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.
<figure><img src="image_41.png" /></figure>

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The NMR assignments for the CTR of XLF, XRCC4, and BRCT1 can be accessed on Biological Magnetic Resonance Data Bank (BMRB) with the following codes: 52387, 52398, and 52399, respectively. The SAXS data for XLF, XRCC4 in complex with BRCTs, and the CTR of XRCC4 can be accessed on Small Angle Scattering Biological Data Bank (SASDBB) with the following codes: SASDU47, SASDU57, and SASDU67, respectively. The previously published structures of proteins were retrieved from
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All the fluorescent microscopy experiments were performed at least two times, all the biochemical assays were carried out at least in duplicate, the cellular assay were carried out at least with six samples, all the NMR experiments were performed once. All attempts at replication were successful. The NMR experiments, each the spectra are recorded as a single independent experiment as common practice in the NMR community with technical replication and good signal to noise ratio. For NMR experiments, each spectrum is recorded as a single independent experiment. The standard deviation presented for all NMR-derived data is a direct propagation of the noise in each individual spectrum.

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## Antibodies

**Antibodies used**
XRCC4 (Creative Biolabs CBMAB-X0197-YC, 1:2000), XLF (Antibodies-Online ABIN2857060, 1:2000) and MLH1 (Abcam ab92312, 1:1000).

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Primary antibodies were used in the study to confirm expression of various gene products. As these are all commercial antibodies, manufacturers' validation statements can be found using the catalog numbers listed.

## Eukaryotic cell lines

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The vendor for the cell line HEK293 Flp-In T-REx is Thermofisher/Invitrogen. Variants were generated in the J. Stark lab: PMID: 35420136, 31753920

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Cell lines tested negative for mycoplasma contamination.

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## Flow Cytometry

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