# Structural basis of Ku-mediated activation of WRN exonuclease activity

Virginie Ropars, Sayma Zahid, Jeanne Chauvat, Ilaria Ceppi, Philippe Frit, Dennis Gomez, Steven W Hardwick, Pierre Legrand, Julien Karazi, Sonia Baconnais, et al.

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Virginie Ropars, Sayma Zahid, Jeanne Chauvat, Ilaria Ceppi, Philippe Frit, et al.. Structural basis of Ku-mediated activation of WRN exonuclease activity. 2026. hal-05528343

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# Structural basis of Ku-mediated activation of WRN exonuclease activity

## Virginie Ropars

Virginie.ROPARS@cea.fr

Institute for Integrative Biology of the Cell https://orcid.org/0000-0002-3372-6030

## Sayma Zahid

University of Leicester https://orcid.org/0000-0002-1354-9209

## Jeanne Chauvat

Université de Toulouse https://orcid.org/0000-0002-2409-6874

## Ilaria Ceppi

Institute of Research in Biomedicine (IRB) https://orcid.org/0000-0001-6496-8983

## Philippe Frit

Université de Toulouse https://orcid.org/0000-0002-6571-8860

## Dennis Gomez

Institute of Pharmacology and Structural Biology IPBS CNRS https://orcid.org/0000-0001-9942-1451

## Steven Hardwick

Department of Biochemistry, University of Cambridge https://orcid.org/0000-0001-9246-1864

## Pierre Legrand

Synchrotron SOLEIL https://orcid.org/0000-0003-2431-2255

## Julien Karazi

Institute for Integrative Biology of the Cell https://orcid.org/0009-0007-5925-7889

## Sonia Baconnais

Institut Gustave Roussy

## Gerard Pehau-Arnaudet

Institut Pasteur

## Sebastien Britton

IPBS https://orcid.org/0000-0002-7008-5316

## Amanda Chaplin

University of Leicester https://orcid.org/0000-0002-9606-8346

## Jean-Baptiste CHARBONNIER

Institute for Integrative Biology of the Cell https://orcid.org/0000-0002-5219-1983

## Petr Cejka

Institute for Research in Biomedicine https://orcid.org/0000-0002-9087-032X

## Patrick Calsou
## Article

### Keywords:

**Posted Date:** February 16th, 2026

**DOI:** https://doi.org/10.21203/rs.3.rs-5965979/v1

**License:** © Ⓟ This work is licensed under a Creative Commons Attribution 4.0 International License.

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**Additional Declarations:** There is **NO** Competing Interest.
Structural basis of Ku-mediated activation of WRN exonuclease activity

Sayma Zahid*¹,², Jeanne Chauvat*³, Ilaria Ceppi*⁴, Floriana Cappiello*#⁹, Benedetta Perdichizzi*#⁹, Philippe Frit³, Dennis Gomez³, Steven W. Hardwick⁵, Pierre Legrand², Julien Karazi¹, Sonia Baconnais⁷, Gérard Pehau-Arnaudet⁸, Sébastien Britton³, Jean-Baptiste Charbonnier¹, Amanda K. Chaplin#⁵,⁶, Pietro Pichierri#⁹, Petr Cejka#⁴, Patrick Calsou #³α, Virginie Ropars #¹α

1. Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France.

2. Synchrotron SOLEIL, l'Orme des Merisiers, Saint Aubin, France.

3. Institut de Pharmacologie et Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, Toulouse, France.

4. Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Faculty of Biomedical Sciences, Bellinzona, Switzerland.

5. Cryo-EM Facility, Department of Biochemistry, University of Cambridge, Sanger Building, Tennis Court Road, Cambridge CB2 1GA, United Kingdom.

6. Leicester Institute for Structural and Chemical Biology, Department of Molecular and Cell Biology, University of Leicester, Leicester, United Kingdom.

7. Genome Integrity and Cancer UMR 9019 CNRS, Université Paris-Saclay, Gustave Roussy 114 rue Edouard Vaillant, 94805 Villejuif, France.

8. UTECH UBI, Institut Pasteur and CNRS UMR 3528, Paris, France.

9. Mechanisms, Biomarkers and Models Section, Genome Stability Group, Department of Environment and Health, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.

<footnote>*: the authors contribute equally to the article</footnote>
#: corresponding authors

α: lead contacts

## ABSTRACT:

Werner (WRN) is the only human RecQ helicase family member with DNA exonuclease activity. WRN promotes genome stability through its functions in DNA replication, repair and telomere maintenance, the deficiency of which presents clinically as Werner syndrome, causing premature aging and cancer predisposition. The main DNA double strand-break sensor Ku70/80 heterodimer (Ku) is a known partner of WRN, which stimulates its nuclease activity. However, the molecular basis of Ku-WRN interplay is currently unknown. Here, we present a high resolution cryo-EM structure of human Ku bound to DNA in complex with the N-terminal WRN exonuclease domain. This structure reveals multiple interaction sites between WRN and the Ku:DNA complex. The catalytic domain of WRN-exo engages with the DNA ends, stabilized by the vWA-like Ku80 domain interacting with the N-terminal APLF-like Ku binding motif (A-KBM) of WRN. Most surprisingly, we visualize the SAP domain of Ku70 stabilized within this complex, and we identify specific contacts mediating this interaction. These interactions were validated by assessing the impact of point mutations on either side of the Ku-WRN interfaces on exonuclease activity with purified recombinant proteins, and on live protein recruitment at biphoton laser-damaged nuclear sites. Finally, we show that disruption of WRN-Ku70 interaction results in aberrant resection of stalled replication forks. Together, we define the architecture of the Ku-WRN exonuclease domain interface and its impact on WRN exonuclease activity, recruitment and replication fork processing.

## INTRODUCTION
Werner (WRN) is a RecQ family protein with 3'-5' helicase and 3'-5' exonuclease activities that play roles in DNA metabolism during replication and repair (1-3). WRN maintains telomere stability and also plays roles at stalled forks to counteract replication stress, thereby ensuring genomic stability (for reviews, (3,4)). Mutations in WRN cause the autosomal-recessive disorder Werner syndrome characterized by premature age-associated diseases including cancer (5,6). WRN function relies in part on its ability to disrupt non-canonical DNA structures including G-quadruplexes (7,8). Recently, targeting WRN helicase activity has emerged as a potential strategy to eradicate tumors with unstable microsatellites that form cruciform structures, the replication of which strictly relies on WRN-mediated unfolding (9).

In large-scale cellular interactome analyses, WRN appeared among the top candidates as partners of the DNA-end sensor Ku, a core protein of the non-homologous end-joining (NHEJ) pathway for the repair of DNA double-strand breaks (DSB) (10-13). WRN has also been shown to interact with the other NHEJ factors including DNA-PKcs (14) and XRCC4-DNA ligase IV complex (15). The Ku interaction with WRN strongly stimulates WRN exonuclease activity in *vitro* (16-19). DNA-PKcs phosphorylates WRN and regulates its enzymatic activities following binding to Ku and DNA ends (14,20). Ku Binding Motifs (KBM) were identified at both the N- and C-termini of WRN via PSI-BLAST analysis (17). Subsequently, it was shown that each of these WRN motifs in isolation is indeed able to bind the Ku heterodimer (21). A recent X-ray structure of the N-terminal KBM of WRN in complex with the vWA domain of *Xenopus laevis* Ku80 (xlKu80) (22) revealed that this WRN-KBM occupies the same site as the KBM of APLF (Aprataxin and PNKP like factor) bound on human Ku heterodimer (23).

Despite the presence of several KBMs in WRN and multiple reports documenting Ku-WRN interaction in cells, disruption of this interaction does not functionally translate into cell sensitivity to ionizing radiation, radiomimetic drugs or topoisomerase II inhibitors (14,24-26). Therefore, WRN plays a marginal, if any role in NHEJ. It has been instead proposed that WRN
prevents unfaithful alternative end-joining of DSBs by limiting MRN-dependent DNA resection at break ends (27). Nevertheless, the precise function of Ku-WRN interaction in cells is unknown.

Ku plays multiple roles outside of NHEJ (28). Among them, a function of Ku at perturbed replication forks is suggested by its presence at collapsed and reversed forks (29-37) and by the sensitivity to replication stress observed upon Ku loss in various organisms (38-40). The NHEJ-independent role of Ku at stressed forks may control resection and ensure proper repair by homologous recombination (reviewed in (29)). Notably, WRN is also enriched at stalled forks (41) and its exonuclease and helicase activities (42-44), as well as non-catalytic functions (45) have been implicated in the resolution of collapsed or reversed replication forks. However, it is not yet known whether WRN function at stressed forks relies on its interaction with Ku.

In this study, we describe a high-resolution cryo-EM structure of human Ku bound to DNA end in complex with the N-terminal exonuclease domain of WRN. This structure highlights the interaction between Ku80 and the N-terminal APLF-like KBM of WRN, while also revealing specific contacts between WRN and the SAP domain of Ku70. To evaluate the functional significance of these interactions, we introduced point mutations at the Ku-WRN interfaces.

We assessed the impact of these mutations on WRN exonuclease activity using purified proteins and on the recruitment of these proteins to biphoton laser-induced nuclear damage sites. Finally, we analyzed how these mutations influence resection at stressed replication forks. Our results decipher the structural basis of the Ku-driven activation of WRN exonuclease activity and argue for its role at stalled replication forks.

## RESULTS
**Structure of the WRN N-terminal exonuclease domain bound to Ku-DNA.** To characterize the interaction between the Ku-DNA complex and the N-terminal region of WRN at the molecular level, we generated a human WRN construct, which encompasses its N-terminal Ku Binding Motif (nA-KBM) and the exonuclease domain, herein termed WRN-exo (Figure 1A). We incubated WRN-exo with full-length human Ku70/80 bound to hairpin DNA (hDNA), which consists of a 14 bp duplex region and a 7 bp duplex region separated by a hairpin with a 4 bp stem and a 5 nt loop (Figure S1A (46)), and prepared cryo-EM grids of this complex sample. We collected data from one of these grids, and obtained a map with an overall resolution of 3.33Å (Figure S1B-G). The map clearly shows that the Ku70/80 heterodimer engages DNA as previously described (46), but additional density could be observed corresponding to WRN-exo and the SAP domain of Ku70 (Figure 1B, S5E). The nA-KBM of WRN can be seen bound to the outer surface of the vWA domain of Ku80, whilst the catalytic exonuclease domain of WRN is positioned on the end of the DNA chain away from the DNA end where a hairpin structure is expected. Finally, the SAP domain of Ku70 can be seen engaged with the DNA substrate and WRN-exo. The molecular details and functional consequences of these specific interactions are assessed in the following sections.

**Biophysical characterization of Ku interaction with WRN A-KBMs.** Our cryo-EM structure of Ku70/80-DNA in complex with WRN-exo clearly shows the nA-KBM binding to the periphery of the vWA domain of Ku80 (Figure 2A, S5E), consistent with the crystal structure of the vWA domain of xIKu80 engaged with the WRN nA-KBM (22). The WRN nA-KBM occupies a hydrophobic groove formed between α4 and α5 of the vWA domain of Ku80, with isoleucine 112 of Ku80 packing against cysteine 15 and methionine 19 of WRN nA-KBM. Other prominent residues at this interaction interface are arginine 13 of WRN nA-KBM packing
against aspartates 106 and 109 of Ku80, and the conserved tryptophan 18 of WRN nA-KBM in
close proximity to glutamine 119 of Ku80 (Figure 2A, S2A).

To assess the importance of the nA-KBM in the interaction between WRN and Ku70/80,
relative to other KBMs of WRN, we used isothermal calorimetry (ITC) with full-length
Ku70/80 (KuFL) protein and various WRN N-terminal (n) and C-terminal (c) peptides which
contained APLF (A)- and/or XLF (X)-like KBMs (WRN nA, WRN cA, WRN cX, and WRN
cAX, respectively) (Figure S2B). WRN nA and cA peptides bound to KuFL with micromolar
affinity: 2.8 ± 0.6 µM and 0.5 ± 0.1 µM, respectively (Figure S2C, D). Indeed, AlphaFold3
(47) predicted a model for cA interaction on Ku80 very similar to the structure we obtained for
nA (Figure S2E-G). No interaction was observed between KuFL and WRN cX peptide,
predicted to be a XLF-like motif that would bind to the inner face of the Ku80 vWA domain
(23). In addition, WRN cAX peptide exhibits micromolar affinity for KuFL, similar to that
measured for WRN cA alone (Figure S2C, D). Thus, the interaction of WRN cAX with Ku in
vitro is primarily driven by the A-KBM motif. The lack of cooperativity between WRN cX-
and cA-KBMs found by ITC is in agreement with previous fluorescence polarization data (17).

We then performed ITC measurements with the N-terminal region of WRN in isolation (WRN-
exo) and the KuFL in complex with hDNA at 1:1.2 ratio. WRN-exo had an affinity for KuFL
(Kd = 2.0 ± 0.55 µM) similar to that of WRN nA (Figure 2B and S2D). When the conserved
tryptophan in WRN nA (WRN W18G) was mutated in the WRN-exo construct, no detectable
dissociation constant was measured, indicating loss of binding affinity (Figure 2B and S2D).

Interaction between Ku80 vWA domain and WRN A-KBMs stimulates WRN exonuclease in
vitro. To assess the functionality of the KBM-dependent WRN-Ku interaction, we set up an
exonuclease assay with purified proteins (Figure S3A) and radiolabeled DNA substrates
(Figure S3B-F), similar to those described previously (16). Under our conditions, full-length human WRN protein exhibited a concentration-dependent 3'-5' exonuclease activity, both on a DNA substrate carrying a single-stranded overhang at the 5' end (31 bp + 19 nt 5' overhang) and on a blunt ended DNA substrate (50 bp) (Figure S3B, S3D-F). This activity relied on the integrity of the WRN exonuclease domain since it was abolished by the E84A mutation in the catalytic pocket (Figure S3B, C). Addition of human full-length Ku promoted the WRN exonuclease activity on the 5'-overhang substrate (Figure 2C, D and Figure S3D), while addition of yeast Ku was strongly inhibitory (Figure 2C, D and Figure S3E), supporting the notion that species-specific interaction underlies the observed stimulatory effect. Human and yeast Ku exhibited similar DNA binding activities as assessed by electromobility shift assay (Figure S3G). In contrast to the stimulation of WRN on the 5'-overhang substrate, human Ku inhibited WRN exonuclease-dependent degradation of the 50 bp-long double blunt DNA (Figure S3F).

Ku binding to DNA ends is oriented with Ku70 outward and Ku80 inward from the break end (48). When superimposed onto models of DNA-PK, our structural data indicate that the interaction between WRN-exo and Ku70/80 positions the exonuclease domain on the internal face of Ku70/80 with respect to the DNA break end; i.e. on the other side than DNA-PK (Figure 1B and S5A). Thus, we hypothesized that the stimulation of WRN exonuclease activity on the 5'-overhang substrate in the presence of Ku70/80 is most likely due to the preferential binding of one Ku dimer at the blunt end of the DNA substrate, subsequently positioning WRN-exo to attack the 3' end of the shorter DNA strand (top strand in Figure 2C). Indeed, when the blunt end of the 5'- protruding substrate was blocked with streptavidin, Ku70/80 was no longer able to stimulate the exonuclease activity of WRN, likely because Ku70/80 was not able to bind the blunt end (Figure S4A, compare middle panel with side panels). Furthermore, when we extended the length of the central duplex DNA region, the stimulatory effect was diminished
(Figure S4A), arguing that DNA end-bound-Ku might need to physically engage WRN bound to the overhang.

Next, we questioned whether the KBM-mediated direct interaction between WRN and Ku70/80 was indeed necessary for the Ku-dependent stimulation of WRN exonuclease activity. Guided by our cryo-EM structural data (Figure 2A), we generated mutations at the interface between WRN and Ku. Specifically, isoleucine 112 of the Ku80 vWA domain was mutated to arginine, a position previously reported to be essential for the interaction with the KBM of APLF (23,49). While the Ku I112R mutant protein bound DNA ends as efficiently as Ku WT (Figure S3G), it was unable to stimulate the exonuclease activity of WRN (Figure 2C, D). Conversely, we disrupted both WRN A-KBMs by mutating the conserved tryptophan in WRN nA and WRN cA to glycine (Figure S3A). While WRN W18G-W1410G double mutant in isolation exhibited exonuclease activity comparable to the WT protein (Figures S3B, C), it was not stimulated by either human Ku, WT or I112R (Figure 2E, F and S4B).

WRN N-terminal A-KBM dictates the Ku-dependent recruitment of WRN-exo domain to DNA damage sites in cells. Based on our structural data, we next assessed the impact of the A-KBM-mediated WRN interaction with Ku70/80 in cells. We analyzed the recruitment of the WRN-exo fragment fused to GFP (GFP-WRN-exo) at laser-induced DNA damage sites. To establish to what extent WRN-exo mobilization to damaged sites was Ku-dependent, we used in-house engineered U2OS cells in which the essential endogenous Ku70 was replaced by Ku70 tagged with a mini-auxin inducible degron (mAID). Within a few hours upon addition of indole-3-acetic acid (auxin, IAA), degradation of mAID-Ku70 occurs accompanied by destabilization of Ku80 (50). Since the rapid recruitment of GFP-WRN-exo to micro-irradiated areas was nearly abolished without Ku, we concluded that it was mainly Ku-dependent (Figures 3A, B). Based on our structural and biochemical data, the impact of specific mutations at the Ku:WRN-
exo interface on Ku-dependent WRN-exo recruitment to DNA damage sites was monitored (Figure 3C, D). Either the Ku80 I112R mutation in the nA-KBM binding site or the W18G mutation in the WRN nA-KBM strongly compromised WRN-exo recruitment to nuclear DNA damage sites (Figure 3C, D). Importantly, recruitment to damaged DNA sites of Ku itself, as measured by recruitment of mCherry-Ku70, was not compromised by the Ku80 I112R mutation, showing that the impairment of WRN-exo recruitment was primarily due to a disruption of the interaction between Ku and WRN (Figure S4C). Altogether, these data indicate a major role of the A-KBM-mediated WRN interaction with Ku for WRN mobilization to damaged DNA and subsequent stimulation of WRN exonuclease activity.

### Ku-dependent stimulation of WRN exonuclease occurs during cryo-EM sample preparation.

A hairpin DNA substrate was used for the preparation of our cryo-EM samples (Figure S1A). We modeled the hairpin region close to the vWA domain of Ku80 based on the crystal structure obtained for Ku-hDNA-pXLF, which used the same hairpin substrate (PDB: 6ERG, (23)) (Figure S5B). The Ku70 subunit is positioned near the blunt DNA end, while the Ku80 subunit is located near the hairpin. In the Ku-hDNA:WRN-exo complex, only the density of the 14 bp duplex was clearly visible (colored in red in Figure S5C). Unlike the Ku-hDNA-pXLF structure, we were unable to resolve the hairpin region of the DNA molecule. In the 3' DNA end closest to the hairpin, three nucleotides were missing, and this end was positioned approximately 15 Å away from the active site of the WRN exonuclease domain (Figure 1B, S5E). We reasoned that the hairpin may have been resected during the mixing phase of purified proteins and DNA prior to cryo-EM sample vitrification. Therefore, we performed the WRN exonuclease assay with WRN-exo alone or in the presence of Ku on the hairpin DNA (Figure S5C, D). Indeed, Ku stimulated WRN exonuclease activity from the 3' to the 5' end of hDNA,
most likely explaining the lack of hairpin detection in the structure we obtained (Figure S5D-E).

*The Ku70 SAP domain stimulates the exonuclease activity of WRN in vitro.* A surprising observation from our cryo-EM structure of the Ku-hDNA:WRN-exo complex was that the SAP domain of Ku70 can be visualized and is in contact with both the DNA substrate and the WRN exonuclease domain (**Figure 1B and S5E**). Amino acid W145 of WRN docks against T577 and K596 of the SAP domain (**Figure 4A, B and S6A**). The residues K596 and T577 of Ku70 involved in these interactions are well conserved (**Figure S6A**). Notably, although the SAP domain of Ku70 was positioned near WRN exonuclease domain by AlphaFold3 as observed in our cryo-EM structure, the predicted SAP domain orientation was different, with no contact between the residue K596 of Ku70 and WRN exonuclease domain (**Figure S6B**).

To assess the functional relevance of the Ku70 SAP domain on WRN function, the exonuclease activity of WRN was first measured in the presence of Ku with both Ku70 and Ku80 C-terminal domains deleted (Ku CC) (**Figure S6C**). We have previously shown that Ku CC is not impaired in DNA binding activity (**Figure S6D**) (23). Unlike with full-length Ku70/80, the addition of the Ku CC construct resulted in no stimulation of WT WRN exonuclease activity, and even a strong inhibition with the A-KBM mutated version of WRN (W18G-W1410G) was observed (**Figure S6E-H**). Next, to rule out any possible impact of the deletion of the Ku80 C-terminal domain, we mutated KuFL at various Ku and WRN residues deduced from the structure of the Ku70 SAP-WRN interface (**Figure 4B**). Although Ku K596A mutant (SAP domain) exhibited DNA binding activity identical to WT Ku (**Figure 4C, D**), it no longer stimulated WRN exonuclease activity (**Figure 4E, F**). Similarly, W145A and R149E WRN-exo mutant proteins were no longer stimulated by WT Ku (**Figure 4G, H**), while they showed identical substrate
degradation activity without Ku (Figure 4I, J). These data show that the interaction of the Ku70 SAP domain with WRN contributes to the stimulation of WRN exonuclease activity *in vitro*.

**The Ku70 SAP domain contributes to WRN recruitment to DNA damage sites.** To validate the key residues identified at the WRN-exo:Ku interface outside the A-KBM in a cellular context, we used the GFP-WRN-exo recruitment assay at laser-damaged nuclear areas in U2OS cells. We then assessed the impact of mutations in either WRN-exo (Figure 5A, B) or Ku subunits (Figure 5C, D, Figure S7D), as guided by our structural data. To ensure the integrity of the NHEJ complex at damaged sites in the presence of Ku mutants, we simultaneously monitored the recruitment of mCherry-PAXX, a Ku partner that was co-expressed within the same cells (Figure 5A-C) (PAXX recruitment at laser-induced DNA damage is strictly Ku-dependent, (51)). Regarding the mutations in Ku80, we monitored the recruitment of mCherry-Ku70 as a control (Figure 5D). While PAXX recruitment was preserved, WRN-exo recruitment was significantly impaired by the E173R and R149E mutations in WRN-exo as well as by mutations in Ku70 SAP domain, with K596A being the most detrimental while supporting normal PAXX recruitment (Figure 5B-D, Figure S7A and S7C). In addition, the K7E mutation in Ku80 impaired GFP-WRN-exo recruitment while preserving Ku recruitment to laser-damaged sites (Figure 5D and S7C). However, the impact of these mutations was less severe than that of the W18G mutation in WRN-exo or the I112R mutation in Ku80, both of which compromising the binding of WRN N-terminal A-KBM to Ku (Figure 3C, D). It should be noted that the K596E and Q597A mutations in Ku70 SAP domain impaired PAXX recruitment (Figure S7B). Since residues 590-604 in SAP domain are also involved in DNA interaction (52,53), this impairment may reflect a disruption of the interaction between Ku and DNA.

**Disruption of Ku-WRN interaction impacts resection at stressed replication forks.**
Since localization of WRN to laser-induced DNA damage depends on a functional Ku70 interaction domain, and since the exonucleolytic activity of WRN has been shown to protect stalled replication forks from degradation under low dose of camptothecin (CPT) (43), we investigated whether WRN-Ku70 interaction was also involved in the WRN recruitment at CPT-induced stalled replication forks. To this end, U2OS WRN KO cells were complemented with plasmids expressing WRN wild type or the R149E mutant (Figure S8A) and were subsequently analyzed for the recruitment at the replication fork by the SIRF (in situ Protein Interaction with Nascent DNA Replication Forks) assay (Figure 6A, see left of Figure S8C for a scheme depicting the method). As expected, WRN was found already localized at DNA replication forks in untreated cells (Figure 6A). Treatment with CPT resulted in enhanced WRN localization at stalled replication forks (Figure 6A). Despite the impaired binding to Ku70, the WRN-R149E mutant was fully proficient in binding to DNA replication forks either in untreated conditions or after treatment with CPT (Figure 6A). Similarly, localization at HU-stalled forks was not affected by the mutation preventing interaction with Ku70.

Given that WRN-Ku interaction stimulates exonuclease activity, which is required to protect reversed forks from being pathologically degraded (43), we used native nascent ssDNA-PLA to monitor the association of WRN with ssDNA exposed at the regressed arm of the reversed fork in cells expressing the Ku70 SAP domain mutant K596A. While SIRF detects proteins associated to nascent DNA at replication fork, native nascent ssDNA PLA can detect protein associated only with exposed ssDNA in the nascent strand as it forms at reversed forks (see scheme on right of Figure S8C). In these cells, endogenous Ku70 was tagged with the mAID degron and, after supplementation with IAA, only the ectopic untagged Ku70 (either WT or mutant) was expressed (Figure S8B). Since the regressed arm of the reversed forks is composed of the two reannealed nascent DNA strands, degradation unveils nascent ssDNA labelled with IdU (see model on right of Figure S8C). Thus, nascent ssDNA (i.e. anti-IdU-WRN) PLA
specifically identifies the fraction of WRN recruited at reversed forks to protect them from
aberrant degradation. As shown in **Figure S8C**, WRN was significantly more localized at sites
of nascent ssDNA after treatment with 50 nM CPT. Expression of the K596A Ku70 protein
resulted in only a minor reduction in the localization of WRN at nascent ssDNA as seen by
number of PLA spots after CPT treatment (**Figure S8C**).

To indirectly estimate if reversed forks were not reduced in Ku70-K596A mutant, we performed
nascent ssDNA PLA with anti-RAD51 antibodies. While RAD51 can be found at parental
ssDNA at the fork or behind it, it can be detected at nascent ssDNA in reversed forks. PLA
evaluating nascent ssDNA-RAD51 interaction was stimulated by CPT treatment and even more
in cells expressing the Ku70 SAP mutant K596A (**Figure S8D**). Consistent with the SIRF data
obtained in cells expressing the R149E WRN mutant, abrogation of the association with Ku70
did not affect WRN localization at stalled forks (**Figure S8E**). Expression of the exo-dead
E84A WRN mutant results in fork degradation even if its interaction with nascent ssDNA is
unaffected (43). Thus, we analyzed if the expression of the R149E WRN mutant was sufficient
to phenocopy the fork degradation-phenotype associated with the exo-dead WRN. DNA fibers
were prepared from U2OS WRN KO cells expressing the wild type or the R149E mutant WRN
protein after sequential labelling of ongoing forks with CldU and IdU (see scheme in **Figure
6B**). As shown in **Figure 6B**, quantification of the IdU tract length, which is shorter under
degradation, indicated that treatment with 50 nM CPT significantly reduced the length of the
IdU tract when the interaction between WRN and Ku70 was perturbed by the R149E mutation
(**Figure 6B**). These data clearly indicate that WRN R149E cannot protect nascent DNA from
degradation, likely because its exonuclease activity fails to be stimulated by WRN.

The IdU tract length in WRN R149E cells was fully recovered by treatment with the MRE11
inhibitor MIRIN, indicating that MRE11 contributes to the nascent DNA degradation. As
expected, the presence of the E84A mutant greatly stimulated fork degradation and the level of
reduction in the IdU tract length in CPT-treated cells was similar between the two WRN variants (Figure 6B). We next analyzed if abrogating the WRN-Ku70 interaction also affected the ability of CPT-perturbed replication forks to restart DNA synthesis by the DNA fiber assay. Since the seminal work of Jasin's lab, it is known that the fork degradation phenotype is uncoupled from the ability to restart replication once the drug that induces fork stalling is washed out because of alternative mechanisms of fork restart (54,55). The U2OS WRN KO cells complemented with the wild type or the R149E mutant WRN protein were labelled as shown in the scheme of Figure 6C and fork recovery was evaluated by quantification of the IdU tract-length observed in dual-labeled replication tracks, and by quantification of the number of stalled forks represented by replication tracks with only the CldU tract. As shown in Figure 6C, IdU tracts were of similar length in restarting forks analyzed from cells expressing the wild type or R149E WRN after CPT treatment. As a control, cells were also treated with 2 mM HU for 2 h, without any significant difference in the length of restarting forks between the wild-type or the WRN-R149E. Consistent with this quantification, also the percentage of stalled forks did not differ between the two genotypes.

Altogether, these results show that abrogation of the WRN-Ku70 interaction does not affect the ability of WRN to be recruited at stalled forks or, more specifically, at the nascent ssDNA formed at reversed forks. However, the interaction with Ku70 is essential for WRN to protect reversed forks from being pathologically degraded by MRE11.

DISCUSSION

The Werner protein contains several KBMs that directly interact with Ku (17) including two APLF-like KBMs in the N-terminal and the C-terminal regions (nA and cA-KBMs), as well as a XLF-like KBM positioned in tandem with the cA-KBM (cX-KBM) (Figure 1A). We report
here the first structure of the interface between the N-terminal region of human WRN including
the nA-KBM and its exonuclease domain, and Ku70/80. Notably, we found that the WRN nA-
KBM interacts with the vWA domain of Ku80 through both backbone and side chain contacts,
providing new insights into the molecular basis of this interaction. Our data include human full-
length Ku and WRN-exo domain, which extends a previous report with the isolated xIKu80
vWA domain complexed with a hWRN nA-KBM peptide (22). Furthermore, our cryo-EM
structure reveals the detailed molecular interaction between the exonuclease domain of WRN
and Ku, with a particular focus on the SAP domain of Ku70, shedding new light on the
specificities of this protein-protein interaction and allowing the exploration of its functional
implications. A key conceptual conclusion was that WRN binds Ku on the end-distal side of
the heterodimer.

*The Ku70 SAP domain stimulates WRN exonuclease activity in vitro by optimizing its positioning towards the substrate.*

In nuclear extracts, Ku was identified as a partner of WRN that had no effect on the ATPase or
helicase WRN activities but stimulated WRN exonuclease activity *in vitro* (16,18). Ku also
broadened WRN's substrate specificity to include DNA containing oxidized purines (19,56)
that could not be removed by WRN alone (57). Deletion experiments mapped the interaction
necessary for this stimulation to Ku70 and the N-terminal region of WRN (19,56). Our study
further unveiled close contacts between the WRN N-terminal region and the Ku70 SAP domain,
as well as with a Ku80 site outside the A-KBM-binding site. Indeed, our experiments with
purified proteins emphasized a key functional role for the Ku70 SAP-WRN N-terminus
interaction in the Ku-mediated stimulation of WRN exonuclease activity. **In addition, we
showed that the integrity of this interaction is necessary for the optimal recruitment of the
WRN-exo fragment to laser-induced DNA damage in cells, but not of full-length WRN to
replication forks. This difference could be explained by the different structures of the DNA**
intermediates, the contribution of additional WRN domains and/or the presence of specific
interaction partners. Our site-specific mutagenesis approach points the K596 position on Ku70
SAP domain, K7 on Ku80 and E173, R149 on WRN as crucial for the functionality of Ku70
SAP:WRN-exo interaction. Our data with purified proteins suggest that the molecular basis of
Ku-mediated WRN exonuclease stimulation may be through favoring the proximity of the
WRN-exo catalytic domain and the 3' DNA end positioned inward relative to Ku. This may
explain why Ku widens WRN exonuclease substrate preference beyond a 3' recessed strand, as
substrates without Ku may be less accessible to the WRN nuclease active site and/or may result
in less processive WRN nuclease activity.

The Ku70 SAP domain controls WRN exonuclease activity at reversed replication forks.

Ku binds collapsed and reversed forks (29-37). WRN is a major Ku interactor in cells (10-13), and it is found among the most enriched proteins at HU-stalled forks (41). WRN helicase activity (42,44), along with a non-catalytic function of WRN (45) have been implicated in the resolution of collapsed or reversed replication forks. Moreover, WRN exonuclease activity restrains fork degradation (43). A recent study reported that rodent cells expressing a Ku80 mutant unable to bind A-KBM failed to process DNA breaks arising from replication stress and suggested that this failure was due to a defect in Ku interaction with WRN (38). Here, we show further that the Ku70-WRN interaction unveiled by our structural data has a functional significance at stalled replication forks. Although impairing Ku70 SAP interaction with WRN did not reduce WRN recruitment at stalled or reversed forks, it resulted in fork degradation to an extent similar to a WRN exonuclease deficient mutant. Altogether, our data show that Ku70 SAP-WRN interaction with WRN and its impact in WRN exonuclease activity is physiologically relevant for the metabolism of stalled replication forks. The Ku-WRN complex, together with other factors (55,58,59), helps protect reversed replication forks against MRE11-mediated degradation (Figure 6D).
Notably WRN can contact the vWA domain of Ku80 through either its N- or C-terminal A-KBM. Furthermore, our ITC and structural data established that the two WRN KBMs exhibit similar affinity and binding modes with Ku80. While their binding to Ku80 defines different positioning of WRN relative to the DNA substrate, only the one corresponding to the cryo-EM structure reported here may be permissive for the stimulation of WRN exonuclease activity. Therefore, the Ku70-WRN N-terminus interaction is likely a key regulator of the balance between both possible orientations of WRN on Ku80. The regulation of balance between each configuration deserves further exploration but may involve for example WRN phosphorylation by the Ku-associated DNA-PKcs that abolishes the Ku-mediated stimulation of WRN exonuclease (20).

## METHODS

### *Cryo-EM sample preparation of Ku-hDNA_WRN-exo and data acquisition.*

KuFL was mixed with a hairpin DNA (hDNA, (46)) and WRN-exo in a 1:1.2:2 ratio. 3 µl of KuFL-hDNA:WRN-exo complex at ~3 mg/ml were applied on Holey Carbon copper grids, Quantifoil R1.2/1.3, 300 mesh, glow discharged for 60 seconds at 15 mA with a PELCO Easiglow (Ted Pella, Inc). The grids were then blotted for 3 seconds at either force -5 or 0 with filter paper and plunge-frozen in liquid ethane using a FEI Vitrobot Mark IV at 4°C with 100% humidity. Data presented here were collected on a Titan Krios in the Department of Biochemistry, University of Cambridge. 4343 movies for the KuFL-hDNA:WRN-exo complex were collected in accurate centering mode using EPU software (Thermo Ficher). All data collection parameters are given in Supplementary Table 3.

### *Isothermal calorimetry measurements.*
Regarding the interaction measurements between KuFL and the WRN peptides, KuFL heterodimer was thawed and dialysed intensively at 4°C against the buffer 20 mM Tris pH 8, 150 mM NaCl and 5 mM B-ME to remove the glycerol. Regarding the interaction measurements between KuFL-DNA with WRN-exo W18G or WRN-exo wild-type, the proteins were freshly purified and then dialysed against the same buffer described previously. KuFL and WRN-exo variants were concentrated using Amicon Ultra-4 concentrators with 30 kDa and 10kDa cut-off membranes respectively. They were centrifuged for 10 minutes, 13,000 rpm at 4°C before measurement. Routinely, KuFL or KuFL-hDNA (ratio 1:1.2) complexes (WT or variants) were injected into the sample cell (volume 2.2 ml) at a concentration of 10 µM (3 mg). Peptides or WRN-exo variants were loaded into the syringe (290 µl) at a concentration of at least 100 µM, a 10-fold higher concentration than the concentration of KuFL. KuFL or KuFL-hDNA present in the sample cell were titrated by automatic injections (30 injections of 10 µl) of the different peptides or WRN-exo variants. The enthalpy ΔH (in kcal.mol⁻¹), the stoichiometry of the reaction and the association constant Ka (in M⁻¹) are obtained by fitting the isotherm titration curve by a one-binding site model using Origin software (Malvern). All binding experiments are performed in duplicate at 25°C.

## DNA exonuclease assays.

DNA exonuclease assays (10 µl volume) were carried out in a reaction buffer containing 40 mM Tris-HCl (pH 8 at 25°C), 4 mM magnesium chloride, 5 mM DTT, 0.1 mg/ml BSA, 1 mM ATP and 0.5 nM ³²P-labelled oligonucleotide-based DNA substrate (in molecules). The reactions were assembled on ice and incubated at 37°C for 1 hour (16). Next, 0.33 µl of 0.5 M EDTA and 0.66 µl of Proteinase K (20.3 mg/ml, Roche) were added to stop the reaction for 30 minutes at 50°C. An equal amount of formamide dye (95% [vol/vol] formamide, 20 mM EDTA and bromophenol blue) was added and samples were heated at 95°C for 4 minutes before
separating them on 15% denaturing polyacrylamide gel (ratio acrylamide:bisacrylamide 19:1; Bio-Rad). After fixing in a solution containing 40% methanol, 10% acetic acid and 5% glycerol, gels were transferred on 3 MM chromatography paper (Whatman), dried, exposed to storage phosphor screens (GE Healthcare) and scanned by Typhoon FLA 9500 (GE Healthcare).

### Multiphoton laser micro-irradiation.

Live cell microscopy and multiphoton laser micro-irradiation were conducted as previously described (23).

### In situ proximity ligation assay (PLA)

Cells were cultured onto 8-well chamber-slides (Millicell Sigma-Aldrich) and treated for 4 hours with 100 µM IAA. Subsequently, cells were labelled with 100 µM IdU and treated or not with 50 nM CPT in presence of IAA for 1 hour. After treatment, cells were preextracted in 0.5% Triton X-100 for 8 minutes on ice, fixed with 3% PFA, 2% sucrose in PBS 1X for 15 minutes at room temperature (RT), permeabilized in 0.25% Triton X-100 for 15 minutes at RT and blocked in blocking buffer. The in-situ proximity ligation assay (PLA) was performed using the NaveniFlex (Navinci diagnostics) Kit with anti-Mouse PLUS and anti-Rabbit MINUS PLA Probes, according to the manufacturer's instructions. To detect proteins, we used rabbit anti-WRN (Abcam, 1:400), mouse anti-IdU antibody (Becton Dickinson, 1:100) and rabbit anti-RAD51 (Abcam AB133534, 1:200) antibodies.

### In situ Protein Interaction with Nascent DNA Replication Forks (SIRF)

Exponential growing cells were seeded onto microscope chamber slide. The day of experiment, cells were incubated with 125 µM 5-ethynyl-2'-deoxyuridine (EdU) for 8 minutes and treated
with 2 mM HU for 2 hours or 50 nM CPT for 1 hour. Afterwards, cells were preextracted in 0.5% TritonX-100 for 8 minutes on ice and fixed with 3% PFA, 2% sucrose in PBS 1X for 15 minutes at RT. Cells were then permeabilized in 0.25% TritonX-100 for 15 minutes at RT. To detect EdU the Click-iT™ EdU Alexa Fluor™ Imaging Kit (Invitrogen) using 5 µM Biotin-Azide was used for 30 minutes at RT. Cells were blocked in blocking buffer (Navinci diagnostics Kit). After washing with PBS, cells were incubated with the following primary antibodies: rabbit anti-WRN (Abcam, 1:400 for mAID cells or 1:2000 for nucleofected cells) and mouse anti-biotin (Abcam, 1:1000).

## DNA fibers analysis

To evaluate fork degradation cells were pulse-labelled with 50 µM CldU and then labelled with 250 µM IdU with or without 50 nM CPT treatment. When indicated, mirin was added together with IdU, as in the experimental scheme. To evaluate fork recovery cells were pulse-labelled with 50 µM CldU and then were treated or not with 50 nM CPT for 1 hour or 2 mM HU for 2 hours. Afterwards, cells were labelled with 250 µM IdU. For immunodetection of labelled tracks, the following primary antibodies were used: rat anti-CldU/BrdU (Abcam AB6326, 1:50) and mouse anti-IdU/BrdU (Becton Dickinson 347580, 1:10). Images were acquired randomly from fields with untangled fibers using Eclipse 80i Nikon Fluorescence Microscope, equipped with a Virtual Confocal (ViCo) system. The length of labelled tracks was measured using the Image-Pro-Plus 6.0 software. A minimum of 30 individual fibers were analyzed for each experiment. In dot plots, the mean of at least three independent experiments is presented. Data from HU-treated cells for fork recovery evaluation derive from a single replicate. The value of the IdU tract length is reported in micrometers.
## Statistical analysis

Experiments shown are representative of at least three independent biological replicates unless otherwise indicated in the figure legend.

Significance for PLA, SIRF and fibers assays was assessed using the built-in tools in Prism 10 (GraphPad Inc.) by One-way Anova with Tukey's test for multiple comparisons. P < 0.05 was considered as significant.

## FIGURE LEGENDS

**Figure 1. Cryo-EM structure of the Ku70/80-hDNA:WRN-exo complex.**

A: Schematic representation of WRN, Ku70 and Ku80 sequence and constructs used in this study. Dashed grey domains are either deleted or not visible in the final structure. The C-terminal and N-terminal APLF-like Ku Binding Motifs are colored in pink.

B: Left, the X-ray crystal structure of Ku70/80 (yellow/green) bound to an hairpin DNA in grey (PDB 1JEY). Right, the cryo-EM structure of Ku70/80-DNA bound to WRN-exo (purple). Top panel shows experimental map as transparent surface. Structural domains are colored as in A.

**Figure 2. Role of WRN A-KBMs for interaction with Ku and for Ku-dependent stimulation of WRN exonuclease activity.**

A: Overall structure of WRN-exo construct (purple) bound to Ku70/80 heterodimer (yellow/green) and DNA (grey). Zoomed in view of the interaction sites between the N-terminal KBM of the WRN-exo construct (nA-KBM in pink) and the vWA domain of Ku80 (green).
**B:** ITC measurements of KuFL-hDNA titrated by WRN-exo (left panel) and WRN-exo W18G (right panel). Top, thermogram. Bottom, enthalpies measurements versus molar ratio.

**C:** Top, schematic of the assay. Red asterisk indicates the position of the radioactive label. Bottom, representative 15% denaturing polyacrylamide gels showing the degradation of a 5' overhang (19 nt/31 bp) DNA by WRN WT in the presence of human Ku WT or I112R mutant, or *S. cerevisiae* yeast Ku (yKu) as indicated.

**D:** Quantification of overall substrate degradation from experiments such as shown in C. Averages shown; n=3 independent experiments; error bars, SEM.

**E:** Top, schematic of the assay. Red asterisk indicates the position of the radioactive label. Bottom, representative 15% denaturing polyacrylamide gels showing the degradation of a 5' overhang (19 nt/31 bp) DNA by WRN W18G-W1410G mutant in the presence of human Ku WT or I112R, or yeast Ku (yKu) as indicated.

**F:** Quantification of overall substrate degradation from experiments such as shown in E. Averages shown; n≥3 independent experiments; error bars, SEM.

**Figure 3. Role of WRN A-KBMs for Ku-dependent recruitment to DNA damage sites.**

**A:** Top, principle of the laser micro-irradiation experiment. U2OS cells engineered for auxin (IAA)-induced Ku70 knockdown and expressing either GFP-tagged WT or mutant WRN-exo, were micro-irradiated with an 800 nm multiphoton laser to generate DSBs in subnuclear areas. Bottom, representative images before and 30 s after irradiation (irradiated areas are indicated by white arrowheads) of nuclei from U2OS expressing GFP-tagged WRN-exo and depleted or not of Ku70 (±Ku). Scale bar: 10 µm.
**B:** Quantification of WT WRN-exo recruitment (fluorescence accumulation) at laser-induced DNA damage sites under Ku+ or Ku- conditions, as indicated. Results are plotted as mean values of 25 nuclei (+Ku) and 47 nuclei (-Ku) ± SEM.

**C.** Top, principle of the laser micro-irradiation experiment. U2OS cells expressing an inducible shRNA against Ku80 responsive to doxycyclin addition, sh-resistant WT or mutant Ku80 and GFP-WRN-exo were micro-irradiated and accumulation of fluorescence was analyzed. Bottom, quantification of WT WRN-exo recruitment (fluorescence accumulation) at laser-induced DNA damage sites. Results are plotted as mean values of 20 nuclei (WT Ku80) and 18 nuclei (I112R mutant) ± SEM.

**D:** Top, principle of the laser micro-irradiation experiment. U2OS cells knocked-out for WRN and expressing WT or mutant GFP-tagged WRN-exo were micro-irradiated and accumulation of fluorescence was analyzed. Bottom, quantification of WT and mutant WRN-exo recruitment (fluorescence accumulation) at laser-induced DNA damage sites. Results are plotted as mean values of 23 nuclei (WT WRN-exo) and 20 nuclei (W18G mutant) ± SEM.

<caption>**Figure 4. Ku70 SAP domain contributes to stimulation of WRN exonuclease by Ku.**</caption>

**A:** View of the interaction between WRN-exo (purple) and the SAP domain of Ku70 (orange) with Ku70/80 core region. The models are fitted in the cryo-EM map.

**B:** Zoom of the interaction between WRN-exo (purple), Ku80 (green) and the SAP domain of Ku70 (orange). The amino acids outlined correspond to the mutated positions in the following experiments.

**C:** Top, schematic of the DNA substrate. Red asterisk indicates the position of the radioactive label. Bottom, representative 4% native polyacrylamide gel showing the electrophoretic
mobility shift of a 5' overhang (19 nt/31 bp) DNA substrate by human Ku WT or K596A mutant.

**D:** Quantification of DNA binding from experiments such as shown in C. Averages shown; n=3 independent experiments; error bars, SEM.

**E:** Top, schematic of the assay. Red asterisk indicates the position of the radioactive label. Bottom, representative 15% denaturing polyacrylamide gel showing the degradation of a 5' overhang (19 nt/31 bp) DNA by WRN WT in the presence of human Ku WT or K596A mutant.

**F:** Quantification of DNA degradation from experiments such as shown in E. Averages shown; n=3 independent experiments; error bars, SEM.

**G:** Top, schematic of the assay. Red asterisk indicates the position of the radioactive label. Bottom, representative 15% denaturing polyacrylamide gel showing the degradation of a 5' overhang (19 nt/31 bp) DNA by WRN-exo WT or W145A or R149E mutant in the presence of human Ku WT.

**H:** Quantification of DNA degradation from experiments such as shown in G. Averages shown; n=3 independent experiments; error bars, SEM.

**I:** Top, schematic of the assay. Red asterisk indicates the position of the radioactive label. Bottom, representative 15% denaturing polyacrylamide gel showing the degradation of a 5' overhang (19 nt/31 bp) DNA by WRN-exo WT or W145A or R149E mutant.

**J:** Quantification of DNA degradation from experiments such as shown in I. Average shown; n=3 independent experiments; error bars, SEM.

**Figure 5.** Ku70 SAP domain contributes to WRN recruitment to DNA damage sites.
**A:** Top, principle of the laser micro-irradiation experiment. U2OS cells knocked-out for WRN and coexpressing WT or mutant GFP-tagged WRN-exo and mCherry-tagged PAXX, were micro-irradiated and accumulation of each fluorescence was analyzed. Bottom, representative images before (upper frames) and 20 s after irradiation (lower frames, a white arrow indicates irradiated area) of nuclei from U2OS WRN KO expressing WT or mutant GFP-WRN-exo. Scale bar: 10 µm.

**B:** Quantification of fluorescence accumulation at laser-induced DNA damage sites of GFP-WRN-exo (left chart) and mCherry-PAXX (right chart) in U2OS WRN KO cells. Results are plotted as mean values of 74 nuclei (WT WRN-exo), 53 nuclei (E173R WRN-exo mutant) and 52 nuclei (R149E WRN-exo mutant) ± SEM.

**C:** Left, principle of the laser micro-irradiation experiment. U2OS cells engineered for auxin (IAA)-induced Ku70 knockdown, rescued with WT or mutant Ku70 and co-expressing GFP-WRN-exo and mCherry-tagged PAXX, were micro-irradiated and accumulation of each fluorescence was analyzed. Right, results are plotted as mean values of 130 nuclei (WT Ku70), 45 nuclei (K596A Ku70 mutant), 43 nuclei (K575E Ku70 mutant) and 44 nuclei (T577A Ku70 mutant) ± SEM.

**D:** Left, principle of the laser micro-irradiation experiment. U2OS cells engineered for Ku80 knockdown upon doxycyclin addition (doxy), rescued with WT or mutant Ku80 and co-expressing GFP-WRN-exo and mCherry-Ku70, were micro-irradiated and accumulation of each fluorescence was analyzed. Right, results are plotted as mean values of 16 nuclei (WT Ku80) and 31 nuclei (K7E Ku80 mutant) ± SEM.

<caption>Figure 6. Impact of Ku-WRN interaction on resection at stalled replication forks.</caption>
**A:** SIRF PLA analysis of WRN localisation at EdU-labelled DNA in U2OS WRN KO cells expressing wild-type or R149E WRN protein. Left, PLA spots derive from at least 450 cells (three independent experiments). Bars represent mean ± S.E.M. Right, representative images (scale bar: 20 µm).

**B:** Top, schematic of DNA fibers assay and IdU tract length analysis in U2OS WRN KO cells expressing wild-type, R149E or E84A WRN protein to assess fork degradation. Bottom left, dot plots show IdU tract lengths (µm) from three independent replicates. Bars represent mean ± S.E.M. Bottom right, representative fibers images (scale bar: 10 µm).

**C:** Top left, schematic of DNA fibers assay and IdU tract length analysis in U2OS WRN KO cells expressing wild-type or R149E WRN protein to assess fork recovery. Top right, representative fibers images (scale bar: 10 µm). Bottom left, dot plots display IdU tract lengths (µm). Bars represent mean ± S.E.M. Bottom right, the percentage value of stalled forks was calculated by measuring the ratio between the number of CldU-labelled fibers and the number of complete fibers (CldU- and IdU-labelled fibers) x 100 (central panel).

**D:** Scheme of the balance between protection and degradation at a reversed fork, including the present report of the Ku-mediated regulation of WRN exonuclease activity.
ACKNOWLEDGMENTS

P.F., D.G., S.B., V.R., J.B.C. and P.Calsou were supported by the French National Research
Agency (ANR-20-CE11-0026). V.R. and J.B.C. thank ANR support ANR-21-CE12-0019,
ANR 23-CE11-0033, ANR-23-CE18. We (P.F., S.B., P.Calsou, J.B.C. and V.R) would also
thank ARC program 2024 (ARCPGA2023120007404_7962) and Inserm PCSI 2024
MSI&WRN (24CP071-00). A.K.C. thanks the Lister Institute of Preventative Medicine Prize
for support of this research. We would also like to thank the Medical Research Council for the
standard research grant (MR/X00029X/1). Research in the Cejka laboratory is funded by the
Swiss National Science Foundation (SNSF) (Grants 310030_207588 and 310030_205199) and
the European Research Council (ERC) (Grant 101018257). **Worldwide Cancer Research
(WCR), grant n. 24-0262 to P.P.** V.R. and J.B.C. thank access to I2BC platforms (Platform for
Interactions of Macromolecules) and French Infrastructure for Integrated Structural Biology
(FRISBI) ANR-10-INBS-0005. P.F., D.G., S.B. and P.Calsou acknowledge the imaging facility
TRI, member of the national infrastructure France-BioImaging infrastructure supported by the
French National Research Agency (ANR-24-INBS-0005 FBI BIOGEN). P.Calsou is a scientist
from INSERM. S.Z was awarded a PhD fellowship by CEA Saclay, the Soleil Synchrotron and
the Ligue Nationale Contre le Cancer (IP/SC-17131).

**Data availability:** Cryo-EM structure and map are deposited at the PDB and EMDB, respectively with the accession codes: 9HZG and EMD-52524. **Source Data are provided with this paper.**
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# SUPPLEMENTARY MATERIALS

## Supplementary figure legends

### Figure S1. Single-particle cryo-EM image processing workflow for the Ku70/80-hDNA:WRN-exo complex.

A: Secondary structure of the hairpin DNA (hDNA) used for cryo-EM experiments.

B: A representative micrograph of Ku70/80-hDNA:WRN-exo complex recorded on a Titan Krios microscope. Scale bar 50 nm

C: Representative 2D class averages of Ku70/80-hDNA:WRN-exo particles. Scale bar 130Å

D: Angular distribution calculated in cryoSPARC for particle projections. Heat map shows number of particles for each viewing angle

E: Gold-standard FSC curves calculated after cryoSPARC non-uniform refinement and viewing distribution plot.

F: Local resolution map of the Ku-hDNA with WRN cryo-EM map. The colours corresponding to each resolution are displayed on the specific key chart below the maps.

G: Cryo-EM image processing workflow used to determine the structure of Ku70/80-hDNA:WRN-exo complex. Schematic representation of particle picking from collected movies using Warp and processing including 2D classification and ab initio reconstruction using cryoSPARC. Upon ab initio reconstructions, multiple rounds of heterogeneous refinement followed. The map from the initial heterogeneous refinement is shown, followed by the final non-uniform (NU) refinement map.

### Figure S2. Interaction measurement and modeling between Ku and WRN KBM peptides.

A: Top, multiple sequences alignment of WRN nA-KBM (Residue range: 1-21) showing the conserved tryptophan at position (Bold and green highlight). Bottom, logo motif of the nA-KBM (Crooks, G.E. et al., WebLogo: a sequence logo generator. Genome Res 14, 1188-90 (2004)).

B: Sequences of peptides used for ITC measurements, corresponding to the three KBM regions of WRN: two APLF-like motifs (nA and cA, pink) and one XLF-like motif (cX, blue).

C: ITC curves of KuFL titrated by WRN nA-KBM, cA-KBM, cX-KBM or cAX-KBM, from left to right.

D: Dissociation constants (Kd) measured by ITC for interactions between human Ku wild-type or Ku variants (ΔSAP or K596A) and various WRN KBM peptides, WRN-exo, or the WRN-exo W18G mutant.

E: Zoom of the interaction between Ku80 (green) and the WRN cA-KBM (pink) obtained from AlphaFold3.

F: Superimposition of the AlphaFold model of Ku80-WRN cA-KBM (light pink) with the Ku80-WRN nA (purple) cryo-EM structure, showing an RMSD of 0.35 Å between cA-KBM and nA-KBM.
**G:** Superimposition of the Xray structure of Ku80-APLF-KBM (blue, PDB: 6ERF) with the Ku80-WRN nA (purple) cryo-EM structure, showing an RMSD of 0.54 Å between nA-KBM and APLF-KBM.

**Figure S3. Measurement of Ku impact on WRN exonuclease activity with purified proteins.**

**A:** WRN variants used in this study. The polyacrylamide gel was stained with Coomassie Brilliant Blue.

**B:** Top, schematic of the assay. Red asterisk indicates the position of the radioactive label. Bottom, representative 15% denaturing polyacrylamide gel showing the degradation of a 5' overhang (19 nt/31 bp) DNA by WRN variants.

**C:** Quantification of overall substrate utilization from experiments such as shown in B. Average shown; n≥3 independent experiments; error bars, SEM.

**D:** Top left, schematic of the substrates. Red asterisk indicates the position of the radioactive label. Bottom left, representative 15% denaturing polyacrylamide gel showing the degradation of differently labelled 5' overhang (19 nt/31 bp) DNA substrates by WRN WT in the absence or presence of human Ku. Top right, quantification of overall 5'-labelled substrate utilization from experiments such as shown on the left. Bottom right, quantification of overall 3'-labelled substrate utilization from experiments such as shown on the left. Average shown; n≥3 independent experiments; error bars, SEM. Statistical analysis (unpaired t-test): \*p = 0.0258, \*\*p = 0.0002.

**E:** Representative 15% denaturing polyacrylamide gel showing the degradation of a 5' overhang (19 nt/31 bp) DNA by WRN WT in the presence of human or yeast Ku. Red asterisk indicates the position of the labeling.

**F:** Top left, schematic of the substrates. Red asterisk indicates the position of the radioactive label. Bottom left, representative 15% denaturing polyacrylamide gel showing the degradation of differently labelled dsDNA (50 bp) substrates by WRN WT in the absence or presence of human Ku. Top right, quantification of overall 5'-labelled substrate utilization from experiments such as shown on the left. Bottom right, quantification of the 3'-labelled substrate from experiments such as shown on the left. Average shown; n=3 independent experiments; error bars, SEM. Statistical analysis (unpaired t-test): ns = 0.1382 (not significant), \*p = 0.0174.

**G:** Representative 4% native polyacrylamide gel showing the electro mobility shift of a 5' overhang (19 nt/31 bp) DNA substrate by the indicated Ku variants. n=2 independent experiments. Red asterisk indicates the position of the radioactive label.

**Figure S4. Measurement of Ku impact on WRN exonuclease activity with purified proteins and live recruitment of WRN-exo to DNA damage sites.**

**A:** Top, schematic of the substrates. Red asterisk indicates the position of the radioactive label. Bottom, representative 15% denaturing polyacrylamide gels showing the degradation of different 5'overhang DNA substrates either with free ends **or streptavidin-blocked 3' end (blue dot)** by WRN WT in the absence or presence of human Ku.

**B:** Top left, schematic of the substrates. Red asterisk indicates the position of the radioactive label. Bottom left, representative 15% denaturing polyacrylamide gel showing the degradation
of differently labelled 5' overhang (19 nt/31 bp) DNA substrates by WRN W18G-W1410G mutant in the presence of human Ku. Right, quantification of overall substrate utilization from experiments such as shown on the left. Average shown; n=3 independent experiments; error bars, SEM. Statistical analysis (unpaired t-test): ns = 0.1636 (not significant), ns = 0.5506 (not significant).

**C:** Top, principle of the laser micro-irradiation experiment. U2OS cells engineered for Ku80 knockdown upon docycyclin addition (doxy), rescued with WT or mutant Ku80 and co-expressing GFP-WRN-exo and mCherry-Ku70, were micro-irradiated and accumulation of each fluorescence was analyzed. Bottom, results are plotted as mean values of 16 nuclei (WT Ku80) and 19 nuclei (I112R mutant) ± SEM.

**Figure S5: Fate of the hairpin DNA in the Ku-hDNA:WRN-exo complex.**

**A:** Superposition of Ku70/80-hDNA:WRN-exo structure onto the monomeric DNA-PK holoenzyme from PDB 6ZHA showing the WRN exonuclease domain (purple) positioned away from the DNA damage site. DNA-PKcs is shown in grey, Ku70/Ku80 in orange and green, respectively.

**B:** Comparison of the solved hairpin DNA structure in Ku70/80-hDNA:WRN-exo with the structure from PDB 6ERG.

**C:** Secondary structure of the hairpin DNA used for the cryo-EM data. The nucleotides colored in gray are not visible in the cryoEM map.

**D:** Top, schematic of the substrate. Red asterisk indicates the position of the radioactive label. Bottom, representative 15% denaturing polyacrylamide gel showing the degradation of the hairpin DNA substrate used in cryoEM by WRN WT or WRN-exo construct in the presence of Ku.

**E:** Segments of the cryo-EM map in different regions fitted with the Ku70/80-hDNA:WRN-exo model. Top left: nA-KBM of WRN (pink) bound to the vWA domain of Ku80. Top right, exonuclease domain of WRN (purple) interacting with Ku80. Bottom left, SAP domain of Ku70 (orange) interacting with the exonuclease domain of WRN (purple). Bottom right, overall view of the WRN exonuclease domain and its active site with hDNA (grey).

**Figure S6: Ku C-ter domains contribute to stimulation of WRN exonuclease by Ku.**

**A:** Consurf models of Ku70/80 and WRN-exo showing the highly conserved residues in brown and variable residues in blue.

**B:** Alphafold3 model of Ku-hDNA:WRN-exo (light color) superimposed to the cryoEM structure. A zoom-in of the Ku70 SAP domain illustrates the disrupted interaction between WRN-exo (purple) and the residue K596 of Ku70 (orange) in the AlphaFold model.

**C:** Schematic representation of Ku70/80 (light orange/green) constructs used in the following experiments.

**D:** Top, schematic of the substrate. Red asterisk indicates the position of the radioactive label. Bottom, representative 4% native polyacrylamide gel showing the electromobility shift of a 5'
overhang (19 nt/31 bp) DNA substrate by the indicated Ku variants. n=3 independent experiments.

**E:** Top, schematic of the assay. Red asterisk indicates the position of the radioactive label. Bottom, representative 15% denaturing polyacrylamide gel showing the degradation of a 5' overhang (19 nt/31 bp) DNA by WRN WT in the presence of human Ku WT or CC mutant.

**F:** Quantification of overall substrate utilization from experiments such as shown in E. Average shown; n≥3 independent experiments; error bars, SEM. Statistical analysis (unpaired t-test): ns = 0.1757 (not significant), **p** = 0.0010.

**G:** Top, schematic of the assay. Red asterisk indicates the position of the radioactive label. Bottom, representative 15% denaturing polyacrylamide gel showing the degradation of a 5' overhang (19 nt/31 bp) DNA by WRN W18G-W1410G mutant in the presence of human Ku WT or CC mutant.

**H:** Quantification of overall substrate utilization from experiments such as shown in G. Average shown; n=4 independent experiments; error bars, SEM. Statistical analysis (unpaired t-test): ns = 0.6032 (not significant), ***p** = 0.0002.

**Figure S7: Live recruitment of WRN-exo to DNA damage sites.**

**A:** Left, principle of the laser micro-irradiation experiment. U2OS cells knocked-out for WRN and coexpressing WT or mutant GFP-tagged WRN-exo and mCherry-tagged PAXX, were micro-irradiated and accumulation of each fluorescence was analyzed. Right, quantification of fluorescence accumulation at laser-induced DNA damage sites of GFP-WRN-exo and mCherry-PAXX in U2OS WRN KO cells. Right: results are plotted as mean values of at least 57 nuclei (WT WRN-exo), 52 nuclei (Q144R mutant) and 55 nuclei (D152R mutant) ± SEM.

**B:** Left, principle of the laser micro-irradiation experiment. U2OS cells engineered for auxin (IAA)-induced Ku70 knockdown, rescued with WT or mutant Ku70 and coexpressing GFP-WRN-exo and mCherry-tagged PAXX, were micro-irradiated and accumulation of each fluorescence was analyzed (Right part). Right, results are plotted as mean values of 130 nuclei (WT Ku70), 44 nuclei (K596E mutant) and 45 nuclei (Q597A mutant) ± SEM.

**C:** Left, principle of the laser micro-irradiation experiment. U2OS cells engineered for Ku80 knockdown upon doxycylin addition (doxy), rescued with WT or mutant Ku80 and expressing GFP-WRN-exo were micro-irradiated and accumulation of fluorescence was analyzed. Right, results are plotted as mean values of 20 nuclei (WT Ku80) and 18 nuclei (K7E mutant) ± SEM.

**D:** Western blotting analysis showing the expression of Ku and WRN proteins. U2OS cells engineered for auxin (IAA)-induced Ku70 knockdown were rescued with WT or mutant Ku70 or Ku80. Equal protein loading was checked using GAPDH antibody as loading control.

**Figure S8. Impact of Ku-WRN interaction on resection at stalled replication forks.**

**A:** Western blotting analysis showing the expression of WRN protein. Nucleofected U2OS WRN -/- cells were evaluated for WRN wild type, WRN R149E or WRN E84A expression. Equal protein loading was checked using Lamin antibody as loading control.

**B:** Western blotting analysis showing the expression of Ku70 protein. U2OS Ku70-WT and U2OS Ku70-K596A cells were treated with IAA for 4 h to replace Ku70-mAID (upper band)
by untagged Ku70 WT or K956A (lower band).. Equal protein loading was checked using
GAPDH antibody as loading control.

C: Top, schematic representation of the difference between Edu-WRN PLA (SIRF WRN) and nascent ssDNA-WRN PLA (native IdU-WRN) assays. In situ PLA between WRN and nascent ssDNA in U2OS cells expressing Ku70-WT or Ku70-K596A. After 4 h IAA treatment, cells were labelled with IdU and treated with or without CPT. Bottom left, PLA spots derive from at least 250 cells (three independent experiments). Bars represent mean ± SEM. Bottom right, representative images (scale bar: 20 µm).

D: Top, in situ PLA between RAD51 and nascent ssDNA in U2OS cells expressing Ku70-WT or Ku70-K596A. After 4 h IAA treatment, cells were labelled with IdU and treated with or without CPT. PLA spots derive from at least 160 cells (three independent experiments). Bars represent mean ± SEM. Bottom, representative images (scale bar: 20 µm).

E: Top, SIRF PLA analysis of WRN localization at EdU-labelled DNA in U2OS cells expressing Ku70-WT or Ku70-K596A. PLA spots derive from at least 530 cells (three independent experiments). Bars represent mean ± SEM. Bottom, representative images (scale bar: 20 µm).

METHODS

*Expression and Purification of WRN-exo.*

The codon-optimized cDNA for expressing the N-terminal region of WRN (1-236) in *E. coli*, referred to as WRN-exo, was cloned into a pET11a vector supplied by Genscript, between the NdeI and BamHI restriction sites. WRN-exo mutants (W18G, R149E and W145A) were prepared by Genscript through mutagenesis of the corresponding pET11a-WRN-exo plasmid. For a final 800 ml culture, 20 ml of pre-culture was diluted in LB culture medium supplemented with 0.2 mg/ml of ampicillin and incubated at 37°C (OD<sub>600nm</sub> of ~0.2). Once OD<sub>600nm</sub> of the cell cultures reached a value between 0.6-0.8, 250 µM of IPTG (final concentration) was supplemented to the medium. The cultures were incubated at 20°C overnight at 180 rpm. The cell cultures were arrested by centrifugation 20 minutes at 3,500 rpm and at 4°C (Beckman JLA 8.1000). The cell pellet of 800 ml of culture was resuspended with 20 ml buffer containing 20 mM Tris-HCl pH 9.0, 150 mM KCl, 850 mM NaCl, 20 mM imidazole pH 9.0, 10 mM β-mercaptoethanol (B-ME) and flash-frozen in liquid-nitrogen for storage at -80°C. The bacterial pellets were resuspended on ice in 35 ml wash/lysis buffer containing 20 mM Tris-HCl, 150 mM KCl, 850 mM NaCl, 20 mM imidazole, 10 mM BME, 5% glycerol, 300 µg lysozyme and cocktail of protease inhibitor EDTA-Free (Roche). Cells were sonicated by ultrasound on ice for 5 cycles of 1 minute with 1 s ON/1 s OFF pulses and an increasing amplitude from 30% to 60%. Then, cells were incubated during 20 minutes on ice with 2 µl of benzonase (25 kU stock, Sigma Aldrich) and 10 mM MgCl<sub>2</sub>. The lysate was then clarified by centrifugation during 30
minutes at 20,000 rpm at 4°C (rotor JA 25.50 on Beckman Coulter). The soluble fraction was
filtered through a 0.45 µm filter to eliminate aggregates before incubation with 20 ml nickel-
coated agarose beads for 1 hour at 4°C on a rotating wheel. The beads were washed with 7
column volumes (CV) of wash/lysis buffer and eluted with 5 times 10 ml (total 2.5 CV) of 20
mM Tris-HCl, 150 mM NaCl, 300 mM imidazole and 10 mM B-ME. Elution fractions of
interest were pooled and dialysed against buffer Q<sub>AWRN</sub> (20 mM Tris-HCl, 50 mM NaCl, 10
mM B-ME and 0.5 mM EDTA) overnight at 4°C using a Spectra-Por (Spectrum<sup>TM</sup>) dialysis
membrane with a cut-off of 6-8 kDa. The dialysed WRN protein was loaded onto the pre-
equilibrated Resource Q column (6 ml, Cytiva). The column was washed with 3 CV of buffer
Q<sub>AWRN</sub>. The elution of WRN-exo was carried out by performing a gradient from 0 to 40% of
buffer containing 20 mM Tris-HCl, 1 M NaCl, 10 mM BME and 0.5 mM EDTA over 12 CV.
The elution fractions of interest were pooled and dialysed extensively against a freezing buffer
(FB) containing 20 mM Tris-HCl, 150 mM NaCl, 5 mM B-ME, 0.5 mM EDTA and 5%
glycerol, using a dialysis membrane with a cut-off of 6-8 kDa at 4°C.

Expression and Purification of recombinant proteins.

For cryoEM, exonuclease assays and ITC, the human full-length Ku70(1-609)/Ku80(1-732) or KuCC (Ku70 (1-544)/Ku80(1-551)) or KuFL-K596A were expressed in *Sf21* insect cells using a MultiBac expression system (1). KuFL-K596A was prepared by Genscript through mutagenesis of the pFL_Ku70_Ku80 plasmid. The Ku80 subunit contains a 10x His-tag followed by a TEV protease site on its N-terminus. The Ku70/Ku80 heterodimer wild-type and variants were purified according to the protocol described in Nemoz et al., 2018 (2). WRN mutants (W18G-W1410G and WRN W18G) were prepared by mutating the respective pFB-MBP-WRN-his plasmid by QuickChange site-directed mutagenesis kit following manufacturer's instructions (Agilent Technology). Primers used for site-directed mutagenesis are listed in Supplementary Table 1. The point mutants were expressed and purified using the same procedure as the wild-type protein (3). Briefly, WRN wild-type and WRN mutants were expressed in *Spodoptera frugiperda* 9 (*Sf9*) insect cells using bac to bac baculovirus expression system (Invitrogen) and purified by first binding to amylose resin followed by the cleavage of maltose-binding protein (MBP) tag with prescission protease and subsequently binding to nickel-NTA agarose resin (Qiagen) followed by dialysis for 2 hours (4). Yeast Ku was expressed in *Sf9* cells and purified by affinity chromatography taking advantage of the Ku70 N-terminal MBP tag and the Ku80 C-terminal FLAG-tag (5).
### DNA substrate preparation.

Oligonucleotide-based DNA substrates were radiolabeled either at the 5' terminus with [γ-³²P]ATP (Perkin Elmer) and T4 polynucleotide kinase (New England Biolabs), or at the 3' terminus with [α-³²P]dCTP (Perkin Elmer) and terminal transferase (New England Biolabs) according to the manufacturer's instructions (3). Unincorporated nucleotides were removed using Micro Bio-Spin P-30 Tris chromatography columns (Biorad). The 5' overhang DNA substrate was prepared by annealing of the oligonucleotides X12-4SC and X12-3, while the dsDNA substrate was prepared by annealing of the oligonucleotides X12-3 and X12-4C. The biotinylated 5' overhang DNA substrate was prepared by annealing oligonucleotides PC216_50nt and PC209. Oligonucleotides used for DNA substrate preparation are listed in Supplementary Table 2.

The hairpin DNA substrate used for cryoEM and ITC was previously described by Walker et al., 2001 (6). The oligonucleotides of 34 and 21 nucleotides were purchased from Eurofins or Eurogentec. Lyophilized oligonucleotides were resuspended in ultrapure water and complementary strands were mixed at a 1:1 molar ratio and a final concentration of 500 µM. Then, the mixture was heated at 90°C for 5 minutes in a microcentrifuge tube and cooled slowly overnight to room temperature.

### CryoEM data acquisition, images processing, refinement and model building.

Data presented here were collected on a Titan Krios in the Department of Biochemistry, University of Cambridge. All data collection parameters are given in Supplementary Table 3. 4343 movies for the KuFL-hDNA:WRN-exo complex were collected in accurate centering mode using EPU software (Thermo Ficher). CTF correction, motion correction, and particle picking were performed using Warp (7). 262,908 particles for KuFL-hDNA:WRN-exo picked by boxnet2 masked neural network model in Warp were imported into CryoSPARC (8) and were subjected to 2D classification, and *ab initio* reconstruction to generate an initial 3D model. Initial 3D models were subjected to multiple rounds of heterogeneous refinement to remove particles which did not represent Ku. The models' resolution and density quality were further improved by performing homogeneous refinement and finally non-uniform refinement. The classification process is summarized schematically in Figure S1G. The reconstruction at 3.33 Å of KuFL-hDNA:WRN-exo contained 83,379 particles. Final map resolutions were calculated in CryoSPARC (7) by Fourier shell correlation at 0.143 cut-off. For structure refinement, previously published crystallographic models of Ku70/80 with or without hDNA (PDB: 1JEQ, 1JEY, 6ERF) were rigid body-fitted into the final cryo-EM maps using UCSF Chimera (9). The
crystallographic models of WRN exonuclease domain (PDB: 2FBT) and the N-terminal KBM of WRN (PDB: 6TYV) were manually adjusted, refined and extra residues were built using Coot (10). Several rounds of real-space refinement were then performed using PHENIX (11) until outliers were fixed.

### Synthetic peptides.

The synthetic peptides containing the KBM motif were purchased from Genecust at 95% purity, and the concentrations of the peptide stock solutions were determined by amino acid composition analyses. WRN peptides used in ITC measurements are listed below:

* pWRNnA Ac-TTAQQRKCPEWMNVQN-NH2

* pWRNcA Ac-SSAERKRRLPVWFAK-NH2

* pWRNcX Ac-SKKLMDKTKRGGLFS-OH

* pWRNcAX Ac-SSAERKRRLPVWFAKGSDTSKKLMDKTKRGGLFS-OH at 25°C.

### Electrophoretic mobility shift assay.

EMSAs were performed in 15 µl volume with the same reaction buffer utilized for the exonuclease assays. After incubation at 37°C for 1 hour, 5 µl of EMSA loading dye (bromophenol blue and 50% glycerol) were added and samples were immediately loaded either on 4% native polyacrylamide gels. Gels were run on ice, transfer on 17 CHR chromatography paper (Whatman), dried, exposed to storage phosphor screens (GE Healthcare) and scanned by Typhoon FLA 9500 (GE Healthcare).

### Cell lines, cell culture.

U2OS cells (human osteosarcoma cell line from a female patient (1) from ECACC, Salisbury, UK) and HEK-293T human embryonic cells, were grown in Dulbecco's modified Eagle's medium (DMEM, Eurobio, France) supplemented with 10% fetal calf serum (Eurobio, France), 125 U/ml penicillin, and 125 µg/ml streptomycin. Cells were maintained at 37°C in a 5% CO₂ humidified incubator.

### Cell engineering.

All oligonucleotides used (Supplementary Table 1) were purchased from Eurofins Genomics (Ebersberg, Germany). Restriction and modifying enzymes (Phusion and T4 DNA Ligase) were
from ThermoFisher Scientific (Illkirch, France). All constructs were checked by sequencing (Eurofins Genomics).

The generation of U2OS cells expressing an inducible shRNA against Ku80 and the generation of U2OS cells expressing a mini-auxin-inducible degron-tagged Ku70 protein (mAID-Ku70) in place of the endogenous Ku70 protein have been previously described (13,14).

U2OS WRN KO cells were constructed by transfection via the CRISPR/Cas9 system using two guide RNAs, WRN-1: 5' AAACCAGTACAGGTGATCTG 5' and WRN-2: 5' ATCCTGTGGAACATACCATG 3' inserted by restriction cloning into the pCAG-eCas9-GFP-U6-gRNA (pCAG-eCas9-GFP-U6-gRNA was a gift from Jizhong Zou (Addgene plasmid # 79145; http://n2t.net/addgene:79145 ; RRID:Addgene_79145) at Bbs1 restriction sites. Cells were transfected according to the manufacturer's instructions using lipofectamine 2000 (Thermo Fisher Scientific).

Production of lentiviral particles in HEK-293T cells and transduction of U2OS and HEK-293T cells were performed as previously described (15).

### Plasmids and DNA manipulations.

The lentiviral vectors allowing expression of mCherry-tagged human PAXX was described (16).

Ku80 expression vectors: the untagged shRNA-resistant Ku80 and the Ku80-I112R lentiviral expression vector were already described (2). The Ku80-K7E mutant expression vector was obtained by overlap PCR mutagenesis from the untagged shRNA-resistant Ku80 vector using Kpn2-Ku80-K7E-F and pLV-R as mutated inner primers. The PCR fragments were then inserted at the Kpn2I-BcuI restriction site of the pLV3 vector backbone.

Ku70 expression vectors: the Ku70-mutant expression vector was obtained by PCR amplification of the Ku70 coding sequence from the previously described pLV3-FLAG-shR-Ku70 vector (14) using Kpn2-Ku70-F and Bcu-Ku70-R as outer primers and Ku70-mut-F and Ku70-mut-R as inner primers. The PCR fragments were then inserted at the Kpn2I-BcuI restriction site of the pLV3 vector backbone using the Hot-Fusion strategy. The expression vector for mCherry-tagged human Ku70 was constructed by PCR amplification of Ku70 cDNA with Mlu-Ku70-F and Bcu-Ku70-R primers and insertion by Hot-Fusion between MluI and BcuI restriction sites downstream the coding sequence of mCherry previously inserted in the pLV3 vector.

WRN expression vectors: the GFP-WRN-exo-mutant expression vector were obtained by PCR amplification of the WRN exonuclease domain coding sequence from the pLV-hWRN-shRes
vector using Kpn2-WRN-F and Mlu-WRN-236-R as outer primers and WRN-mut-F and WRN-mut-R as mutated inner primers. The PCR fragments were then inserted at the Kpn2I-MluI restriction sites downstream the coding sequence of GFP previously inserted in the pLV3 vector.

The pLV-hWRN-shRes vector was obtained by PCR amplification of the human WRN cDNA (kind gift of Dr Karlseder, (17)) using hWRN-Mlu-F and hWRN-Xma-R primers. The resulting PCR fragment was then inserted at the MluI-XmaI restriction site in the previously described pLV-tTR-KRAB vector (2).

## Nucleofection

KO WRN U2OS cells (U2OS WRN KO) were transiently nucleofected with pLVX-WRN wild-type, pLVX-WRN R149E and pLVX-WRN E84A plasmids. 10 µg of DNA were used for 1.5 × 10⁶ cells, with 2 pulses of 950 V lasting 30 ms by Invitrogen Neon Transfection system (Invitrogen). Afterwards, cells were seeded in 10% FBS medium to carry out the different experimental assays.

## Western blotting analysis

Western blotting analysis was performed using standard methods. Blots were incubated with primary antibodies against LAMIN B1 (Abcam AB16048, 1:20000), WRN (Abcam AB200, 1:2000), GAPDH (Millipore mab374, 1:10000), Ku70 (Santa Cruz Biotechnology ab92450, 1:1000).

After incubations with horseradish peroxidase-linked secondary antibodies (Jackson immunosciences), the blots were developed using the chemiluminescence detection kit ECL-Plus (Amersham) according to the manufacturer's instructions.
<table><caption>Supplementary Table 1. Oligonucleotides used for site-directed mutagenesis of WRN and Ku constructs, and CRISPR guide for WRN</caption><thead><tr><td>Oligonucleotide Name</td><td>Primer Sequence (5' to 3')</td></tr></thead><tbody><tr><td>WRN_W18G_F</td><td>GCACAGCAGCGGAAATGTCCTGAAGGGATGAATGTGCAGAATAAAAGATG</td></tr><tr><td>WRN_W18G_R</td><td>CATCTTTTATTCTGCACATTCATCCCTTCAGGACATTTCCGCTGCTGTGC</td></tr><tr><td>WRN_W1410G_F</td><td>GAGAAAGAGACGATTACCTGTGGGGTTTGCCAAAGGAAGTGATACCAG</td></tr><tr><td>WRN_W1410G_R</td><td>CTGGTATCACTTCCTTTGGCAAACCCCACAGGTAATCGTCTCTTTCTC</td></tr><tr><td>Guide CRISPR WRN n°2</td><td>AAACCAGTACAGGTGATCTG</td></tr><tr><td>Guide CRISPR WRN n°4</td><td>ATCCTGTGGAACATACCATG</td></tr><tr><td>Kpn2-Ku70-F</td><td>CCTCGAGGTTTAAACTACGGgatcTCCGGAgccacc</td></tr><tr><td>Mlu-Ku70-F</td><td>CGGCATGGACGAGCTGTACAAGACGCGTATGTCAGGGTGGGAGTCATATTAC AAAACC</td></tr><tr><td>Bcu-Ku70-R</td><td>CCAGAGGTTGATTATCATATGACTAGTTCAGTCCTGGAAGTGCTTGGTGAGGG CTTC</td></tr><tr><td>Ku70-K575E-F</td><td>CAGCAAGGGTACGCTGGGCgAGTTCACTGTGCCCCATGCTGAAAG</td></tr><tr><td>Ku70-K575E-R</td><td>CTTTCAGCATGGGCACAGTGAACTcGCCCAGCGTACCCTTGCTG</td></tr><tr><td>Ku70-T577A-F</td><td>GGGTACGCTGGGCAAGTTCgCTGTGCCCCATGCTGAAAGAGGCCTGCC</td></tr><tr><td>Ku70-T577A-R</td><td>CATGGGCACAGcGAACTTGCCCAGCGTACCCTTGCTGATG</td></tr><tr><td>Ku70-K596A-F</td><td>CTTACGGGCTGAAGAGTGGgCTGAAGgcGCAGGAGCTGCTGGAAGCCCTC</td></tr><tr><td>Ku70-K596A-R</td><td>GAGGGCTTCCAGCAGCTCCTGCgcCTTCAGcCCACTCTTCAGCCCGTAAG</td></tr><tr><td>Ku70-K596E-F</td><td>CTTACGGGCTGAAGAGTGGgCTGAAGgAGCAGGAGCTGCTGGAAGCCCTC</td></tr><tr><td>Ku70-K596E-R</td><td>GAGGGCTTCCAGCAGCTCCTGCTcCTTCAGcCCACTCTTCAGCCCGTAAG</td></tr><tr><td>Ku70-Q597A-F</td><td>CGGGCTGAAGAGTGGgCTGAAGAAGgcGGAGCTGCTGGAAGCCCTCACC</td></tr><tr><td>Ku70-Q597A-R</td><td>GGTGAGGGCTTCCAGCAGCTCCgcCTTCTTCAGcCCACTCTTCAGCCCG</td></tr><tr><td>Kpn2-WRN-F</td><td>ccactgTCCGGAACCGCCGCCATGAGTGAAAAAAAATTGG</td></tr><tr><td>Mlu-WRN-236-R</td><td>cctcttACGCGTCCTTTGCACAGTATCATCCAAAATCTC</td></tr><tr><td>Kpn2-Ku80-K7E-F</td><td>ctcgtcTCCGGAATGGTGCGGTCGGGGAATgAGGCAGCTGTTGTGCTGTG</td></tr><tr><td>pLV-R</td><td>CCAGTCAATCTTTCACAAATTTTGTAATCCAGAGG</td></tr><tr><td>WRN-Q144R-F</td><td>GTAGGAATTGAAGGAGATCgGTGGAAACTTCTACGAGACTTTGATATC</td></tr><tr><td>WRN-Q144R-R</td><td>CAAAGTCTCGTAGAAGTTTCCACcGATCTCCTTCAATTCCTACACC</td></tr><tr><td>WRN-R149E-F</td><td>CAGTGGAAACTTCTAgAGACTTTGATATCAAATTGAAGAATTTTGTGGAG</td></tr></tbody></table>
<table><caption>**Supplementary Table 2. Oligonucleotides used for DNA substrate preparation. [TB]**
indicates the position of the biotin.</caption><tr><td>WRN-R149E-R</td><td>CTTCAATTTGATATCAAAGTCTtcTAGAAGTTTCCACTGATCTCCTTCAATTCC</td></tr><tr><td>WRN-R173E-F</td><td>GCTGAAATGTACAcgGACCTGGAGCCTTAACAGTCTGGTTAAAC</td></tr><tr><td>WRN-R173E-R</td><td>CTGTTAAGGCTCCAGGTCcgTGTACATTTCAGCTTTTTATTGGC</td></tr><tr><td>WRN-D152R-F</td><td>CTTCTACGAGACTTTcgTATCAAATTGAAGAATTTTGTGGAGTTGAC</td></tr><tr><td>WRN-D152R-R</td><td>CTTCAATTTGATAcgAAAGTCTCGTAGAAGTTTCCACTGATC</td></tr><tr><td>hWRN-Mlu-F</td><td>CTCTCACGCGTACCGCCGCCATGAGTGAAAAAAAATTGGAAACAACTGC</td></tr><tr><td>hWRN-Xma-R</td><td>CTCTGCCCGGGTTAACTAAAAAGACCTCCCCTTTTCGTTTTGTCC</td></tr></table>

<table><thead><tr><td>Name</td><td>Sequence (5' to 3')</td></tr></thead><tbody><tr><td>X12-4SC</td><td>GCGATAGTCTCTAGACAGCATGTCCTAGCAA</td></tr><tr><td>X12-3</td><td>GACGTCATAGACGATTACATTGCTAGGACATGCTGTCTAGAGACTATCGC</td></tr><tr><td>X12-4C</td><td>GCGATAGTCTCTAGACAGCATGTCCTAGCAATGTAATCGTCTATGACGTC</td></tr><tr><td>PC216_50nt</td><td>GTAAGTGCCGCGGTGCGGGTGCCAGGGCGTGCCCTTGGGCTCCCCGGGCG</td></tr><tr><td>PC209</td><td>GATGCATGAGGTGGAGTACGCGCCCGGGGAGCCCAAGGGCACGCCCTGGCACCCG CACCGCGGCACT[TB]AC</td></tr></tbody></table>
<table><caption>Supplementary Table 3. Cryo-EM data collection, refinement and validation statistics</caption><thead><tr><th colspan="2">Ku70/80-hDNA-WRN-exo</th></tr><tr><td colspan="2">PDB:9HZG</td></tr><tr><td colspan="2">EMD-52524</td></tr></thead><tbody><tr><td colspan="2"><strong>Data collection and processing</strong></td></tr><tr><td>Detector</td><td>Gatan K3</td></tr><tr><td>Magnification</td><td>130k</td></tr><tr><td>Energy filter slit width (eV)</td><td>20</td></tr><tr><td>Voltage (kV)</td><td>300</td></tr><tr><td>Flux on detector (e/pix/sec)</td><td>12.475</td></tr><tr><td>Electron exposure on sample (e- /Å<sup>2</sup>)</td><td>47.24</td></tr><tr><td>Target defocus range (μm)</td><td>0.7-2.5</td></tr><tr><td>Calibrated pixel size (Å)</td><td>0.652</td></tr><tr><td>Symmetry imposed</td><td>C1</td></tr><tr><td>Extraction box size (pixels)</td><td>400</td></tr><tr><td>Initial particle images (no.)</td><td>262908</td></tr><tr><td>Final particle images (no.)</td><td>83379</td></tr><tr><td colspan="2"><strong>Refinement</strong></td></tr><tr><td>Map resolution at FSC=0.143 (Å)*</td><td>3.33</td></tr><tr><td>Model composition</td><td></td></tr><tr><td>Non-hydrogen atoms</td><td>10561</td></tr><tr><td>Protein residues</td><td>1302</td></tr><tr><td>Nucleotides</td><td>34</td></tr><tr><td>B factor (Å<sup>2</sup>)</td><td></td></tr><tr><td>Protein</td><td>123.63</td></tr><tr><td>DNA</td><td>224.58</td></tr><tr><td>R.m.s deviations</td><td></td></tr><tr><td>Bond lengths (Å)</td><td>0.003</td></tr><tr><td>Bond angles (°)</td><td>0.488</td></tr><tr><td>Validation</td><td></td></tr><tr><td>Molprobity score</td><td>2.37</td></tr><tr><td>Clashscore</td><td>31.91</td></tr><tr><td>Poor rotamers (%)</td><td>0.3</td></tr><tr><td>Ramachandran plot</td><td></td></tr><tr><td>Favored (%)</td><td>94.34</td></tr><tr><td>Allowed (%)</td><td>5.35</td></tr><tr><td>Disallowed (%)</td><td>0.31</td></tr></tbody></table>
SUPPLEMENTARY BIBLIOGRAPHY

1. Gorda, B., Toelzer, C., Aulicino, F. and Berger, I. The MultiBac BEVS: Basics, applications, performance and recent developments. *Methods Enzymol*, **660**, 129-154 (2021).

2. Nemoz, C., Ropars, V., Frit, P., Gontier, A., Drevet, P., Yu, J., Guerois, R., Pitois, A., Comte, A., Delteil, C. *et al*. XLF and APLF bind Ku80 at two remote sites to ensure DNA repair by non-homologous end joining. *Nature Structural & Molecular Biology*, **25**, 971-980 (2018).

3. Pinto, C., Kasaciunaite, K., Seidel, R. and Cejka, P. Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases. *eLife*, **5** (2016).

4. Reginato G, Cannavo E, Cejka P. Physiological protein blocks direct the Mre11-Rad50-Xrs2 and Sae2 nuclease complex to initiate DNA end resection. *Genes Dev.* **31**(23-24):2325-2330 (2017).

5. Mengoli V, Ceppi I, Sanchez A, Cannavo E, Halder S, Scaglione S, Gaillard PH, McHugh PJ, Riesen N, Pettazzoni P, Cejka P. WRN helicase and mismatch repair complexes independently and synergistically disrupt cruciform DNA structures. *EMBO J*, **42**(3):e111998. doi: 10.15252/embj.2022111998. Epub 2022 Dec 21 (2023).

6. Walker, J.R., Corpina, R.A. and Goldberg, J. Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair. *Nature*, **412**, 607-614 (2001).

7. Tegunov D, Cramer P. Real-time cryo-electron microscopy data preprocessing with Warp. *Nat Methods*. **16**(11):1146-1152 (2019).

8. Punjani, A., Rubinstein, J.L., Fleet, D.J. and Brubaker, M.A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. *Nature methods*, **14**, 290-296 (2017).

9. Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C. and Ferrin, T.E. UCSF Chimera—a visualization system for exploratory research and analysis. *Journal of computational chemistry*, **25**, 1605-1612 (2004).

10. Lohkamp, B., Scott, W. and Cowtan, K. Features and development of Coot. *Acta Crystallographica. Section D: Biological Crystallography*, **66** (2010).

11. Afonine, P.V., Poon, B.K., Read, R.J., Sobolev, O.V., Terwilliger, T.C., Urzhumtsev, A. and Adams, P.D. Real-space refinement in PHENIX for cryo-EM and crystallography. *Acta Crystallographica Section D: Structural Biology*, **74**, 531-544 (2018).

12. Cooper MP, Machwe A, Orren DK, Brosh RM, Ramsden D, Bohr VA. Ku complex interacts with and stimulates the Werner protein. *Genes Dev.* **14**(8):907-12 (2000).

13. Seif-El-Dahan, M., Kefala-Stavridi, A., Frit, P., Hardwick, S.W., Chirgadze, D.Y., Maia De Oliviera, T., Andreani, J., Britton, S., Barboule, N., Bossaert, M. *et al*. PAXX binding to the NHEJ machinery explains functional redundancy with XLF. *Science Advances*, **9**, eadg2834 (2023).

14. Kefala Stavridi, A., Gontier, A., Morin, V., Frit, P., Ropars, V., Barboule, N., Racca, C., Jonchhe, S., Morten, Michael J., Andreani, J. *et al*. Structural and functional basis of inositol hexaphosphate stimulation of NHEJ through stabilization of Ku-XLF interaction. *Nucleic Acids Research*, **51**, 11732-11747 (2023).

15. Cheng, Q., Barboule, N., Frit, P., Gomez, D., Bombarde, O., Couderc, B., Ren, G.-S., Salles, B. and Calsou, P. Ku counteracts mobilization of PARP1 and MRN in chromatin damaged with DNA double-strand breaks. *Nucleic Acids Research*, **39**, 9605-9619 (2011).

16. Frit, P., Amin, H., Zahid, S., Barboule, N., Hall, C., Matharu, G., Hardwick, S.W., Chauvat, J., Britton, S., Chirgadze, D.Y. *et al*. DNA polymerase Lambda is anchored within the NHEJ synaptic complex via Ku70/80. *bioRxiv*, 2024.2008.2012.607588 (2024).

17. Crabbe, L., Verdun, R.E., Haggblom, C.I. and Karlseder, J. Defective telomere lagging strand synthesis in cells lacking WRN helicase activity. *Science*, **306**, 1951-1953 (2004).
1. Pontén, J. and Saksela, E. (1967) Two established in vitro cell lines from human mesenchymal tumours. *Int J Cancer*, **2**, 434-447.
A

<figure><img src="image_2.png" /></figure>

B

<figure><img src="image_3.png" /></figure>

Ku70/80:hDNA
(PDB:1JEY)

<figure><img src="image_4.png" /></figure>

Ku70/80:hDNA:WRN-exo
(This study)
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>WRN-exo W18G</figcaption></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>
<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

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

<figure><img src="image_20.png" /></figure>

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

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

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

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

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

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

<figure><img src="image_27.png" /><figcaption>Figure 4</figcaption></figure>
A

<figure><img src="image_28.png" /></figure>

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

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

C

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

D

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

B

<figure><img src="image_33.png" /></figure>

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

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

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

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

<figure><img src="image_38.png" /><figcaption>Figure 5</figcaption></figure>
<figure><img src="image_39.png" /><figcaption>U2OS WRN KO + WRN-WT</figcaption></figure>

<figure><img src="image_40.png" /><figcaption>U2OS WRN KO + WRN-R149E</figcaption></figure>

<figure><img src="image_41.png" /><figcaption>U2OS WRN KO + WRN</figcaption></figure>

<figure><img src="image_42.png" /></figure>

<figure><img src="image_43.png" /></figure>

<figure><img src="image_44.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_45.png" /></figure>

<figure><img src="image_46.png" /><figcaption>U2OS WRN KO + WRN</figcaption></figure>

<figure><img src="image_47.png" /><figcaption>D</figcaption></figure>

BRCA2
WRNIP1
RAD52
**WRNexo/Ku**
...
**PROTECTION**

<figure><img src="image_48.png" /></figure>

MRE11
...
**DEGRADATION**

<caption>Figure 6</caption>
<figure><img src="image_49.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_50.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_51.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_52.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_53.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_54.png" /><figcaption>Example 2D Classes</figcaption></figure>

<figure><img src="image_55.png" /><figcaption>F</figcaption></figure>

PDB: 9HZG
EMD-52524

Figure S1
A

<table><thead><tr><th></th><th></th><th>WRN N-term A-KBM (nA-KBM)</th></tr></thead><tbody><tr><td>HUMAN</td><td>M</td><td>SEKKLETTAQQRKCPWMNV</td></tr><tr><td>MOUSE</td><td>METTSLQRKFPEWM</td><td>16</td></tr><tr><td>XENLA</td><td>MTSLLQRKLPWMSV</td><td>14</td></tr><tr><td>DANRE</td><td>MGDRTLPKWMNN</td><td>12</td></tr><tr><td>CHICK</td><td>MLKVARPGNTNVDKMSKVDLQDKYPQMCA</td><td>30</td></tr></tbody></table>

<figure><img src="image_56.png" /></figure>

B

<table><thead><tr><th>Peptide</th><th>Sequences</th><th>Residue range</th></tr></thead><tbody><tr><td>pWRN nA (16 Aa)</td><td>Ac-TTAQQRKCPWEWMNVQN-NH<sub>2</sub></td><td>8-23</td></tr><tr><td>pWRN cA (15 Aa)</td><td>Ac-SSAERKRRLPVWFAK-NH<sub>2</sub></td><td>1399-1413</td></tr><tr><td>pWRN cX (15 Aa)</td><td>Ac-SKKLMDKTKRGGLFS-OH</td><td>1418-1432</td></tr><tr><td>pWRN cAX (34 Aa)</td><td>Ac-SSAERKRRLPVWFAKGSDTSKKLMDKTKRGGLFS-OH</td><td>1399-1432</td></tr></tbody></table>

C

<figure><img src="image_57.png" /></figure>

<figure><img src="image_58.png" /></figure>

<figure><img src="image_59.png" /></figure>

<figure><img src="image_60.png" /></figure>

D

<table><thead><tr><th></th><th>ITC cell</th><th>ITC syringe</th><th>Kd (µM)</th></tr></thead><tbody><tr><td>1</td><td>KuFL</td><td>WRN nA</td><td>2.8 ± 0.6</td></tr><tr><td>2</td><td>KuFL</td><td>WRN cA</td><td>0.5 ± 0.1</td></tr><tr><td>3</td><td>KuFL</td><td>WRN cX</td><td>NI</td></tr><tr><td>4</td><td>KuFL</td><td>WRN cAX</td><td>1.3 ± 0.3</td></tr><tr><td>Fig 2B</td><td>KuFL-hDNA</td><td>WRN-exo</td><td>2.0 ± 0.55</td></tr><tr><td>Fig 2B</td><td>KuFL-hDNA</td><td>WRN-exo W18G</td><td>NI</td></tr></tbody></table>

E

<figure><img src="image_61.png" /></figure>

F

<figure><img src="image_62.png" /></figure>

G

<figure><img src="image_63.png" /></figure>

RMSD 0.353Å

RMSD 0.539Å

<caption>Figure S2</caption>
A

<figure><img src="image_64.png" /></figure>

B

<figure><img src="image_65.png" /></figure>

<figure><img src="image_66.png" /></figure>

C

<figure><img src="image_67.png" /></figure>

<figure><img src="image_68.png" /></figure>

D

<figure><img src="image_69.png" /></figure>

<figure><img src="image_70.png" /></figure>

<figure><img src="image_71.png" /></figure>

F

<figure><img src="image_72.png" /></figure>

<figure><img src="image_73.png" /></figure>

<figure><img src="image_74.png" /></figure>

E

<figure><img src="image_75.png" /></figure>

<figure><img src="image_76.png" /></figure>

G

<figure><img src="image_77.png" /></figure>

<figure><img src="image_78.png" /></figure>
<figure><img src="image_79.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_80.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_81.png" /></figure>

<figure><img src="image_82.png" /></figure>

<figure><img src="image_83.png" /></figure>
A

<figure><img src="image_84.png" /></figure>

<figure><img src="image_85.png" /></figure>

Damaged
DNA end

<figure><img src="image_86.png" /></figure>

B

<figure><img src="image_87.png" /></figure>

Ku70/80 in complex with
hairpin DNA (6ERG)

<figure><img src="image_88.png" /></figure>

Ku70/80:WRN-exo
(This study)

C

<figure><img src="image_89.png" /></figure>

D

<figure><img src="image_90.png" /></figure>

E

<figure><img src="image_91.png" /></figure>

WRN nA-KBM

Ku80

<figure><img src="image_92.png" /></figure>

Ku80

WRNexo

<figure><img src="image_93.png" /></figure>

Ku70-SAP

WRNexo

<figure><img src="image_94.png" /></figure>

WRNexo
A

<figure><img src="image_95.png" /></figure>

B

<figure><img src="image_96.png" /></figure>

C

<figure><img src="image_97.png" /></figure>

E

<figure><img src="image_98.png" /></figure>

G

<figure><img src="image_99.png" /></figure>

D

<figure><img src="image_100.png" /></figure>

F

<figure><img src="image_101.png" /></figure>

H

<figure><img src="image_102.png" /></figure>
A

<figure><img src="image_103.png" /></figure>

GFP-WRN-exo

<figure><img src="image_104.png" /></figure>

mCh-PAXX

<figure><img src="image_105.png" /></figure>

B

<figure><img src="image_106.png" /></figure>

GFP-WRN-exo

<figure><img src="image_107.png" /></figure>

mCh-PAXX

<figure><img src="image_108.png" /></figure>

C

<figure><img src="image_109.png" /></figure>

GFP-WRN-exo

<figure><img src="image_110.png" /></figure>

D

U2OS cells

complementing Ku70

complementing Ku80

WRN

Ku70

GAPDH

shKu80

/

WT

/

K7E

mAID-Ku70 + TIR1

/

WT

T577A

K596A

K575E

<figure><img src="image_111.png" /><figcaption>Figure S7</figcaption></figure>
A

U2OS WRN KO

<figure><img src="image_112.png" /></figure>

C

<figure><img src="image_113.png" /></figure>

EdU-WRN PLA (SIRF)

<figure><img src="image_114.png" /></figure>

B

<figure><img src="image_115.png" /></figure>

<figure><img src="image_116.png" /></figure>

Nascent ssDNA-WRN PLA
 native IdU-WRN)

<figure><img src="image_117.png" /></figure>

<figure><img src="image_118.png" /></figure>

D

PLA RAD51-ssDNA

<figure><img src="image_119.png" /></figure>

E

SIRF EdU-WRN

<figure><img src="image_120.png" /></figure>
Supplementary Files

This is a list of supplementary files associated with this preprint. Click to download.

* D1292144072valreportfullP1v2.pdf

* ReportingSummary.pdf

* SourceData.xlsx