Cryo-EM structures of NHEJ assemblies with nucleosomes

Domain: chemistry
Published: 2025-12-24
Editorial outcome: major_revision

Abstract
DNA double-strand breaks (DSBs) are highly deleterious lesions that can trigger cell death or carcinogenesis if unrepaired or misrepaired. In mammals, most DSBs are repaired by non-homologous end joining (NHEJ), which begins when Ku70/80 binds DNA ends and recruits DNA-PKcs to form the DNA-PK holoenzyme. Although recent cryo-EM studies have resolved several NHEJ assemblies, how these factors access DSBs within nucleosomes remains unclear. Here, we present cryo-EM structures of human Ku70/80 and DNA-PK bound to nucleosomes. Ku70/80 binds the DNA end and bends it away from the nucleosome core, while the Ku70 C-terminal SAP domain makes an additional, specific DNA contact. Our DNA-PK–nucleosome structure further reveals the opening of the Ku80 vWA domain, and we show that non-hydrolysable ATP promotes synapsis by stabilising the Ku80-mediated DNA-PK dimer. These structures reveal a model for DSB recognition on nucleosomal DNA and provide insights relevant to targeting NHEJ in cancer therapy.

Introduction
During the NHEJ DNA-repair mechanism, the DNA break is recognised by the Ku70/80 heterodimer, which subsequently recruits DNA-PKcs to form the DNA-dependent protein kinase (DNA-PK) holoenzyme complex at the DNA damage site. It has been shown through recent cryo-EM structures and single-molecule experiments that following assembly of the DNA-PK holoenzyme, a long-range synaptic complex (LRC) is assembled on the broken DNA ends. There are at least two forms of LRCs, composed of dimers of DNA-PK, either mediated by XLF or the C-terminus of Ku80 1–4. These DNA-PK LRCs both hold the DNA ends in a pre-synaptic state at ~115 Å apart. The DNA ends may then require further processing by nucleases and polymerases to allow them to be compatible for ligation. It has recently been shown through mutational analysis that the two LRC DNA-PK dimers have unique roles in either promoting fill-in end processing or DNA end resection 5. Finally, DNA-PKcs is phosphorylated and removed from the complex, allowing for a short-range synaptic complex (SRC) to be formed, where the Ligase IV catalytic domain can engage and ligate the DNA ends, thus completing the DNA repair process 3.

All structures of the NHEJ machinery to date have been solved with “free” short pieces of linear double-stranded DNA. However, in eukaryotes, DNA in the nucleus is compacted and packaged within chromatin, where nucleosomes are the fundamental structural unit. Nucleosomes comprise two copies of each histone H2A, H2B, H3 and H4, together assembling a histone octamer with a basic outer surface, which organises 147 bp of DNA into 1.65 turns of a left-handed superhelix, forming the nucleosome core particle (NCP) 6. Nucleosomes array on eukaryotic genomes like beads on a string, and DSBs will result in chromatinized DNA ends. Several studies have identified PARP-dependent chromatin remodelling as stimulating the early steps of NHEJ, including Ku70/80 loading, suggesting that chromatin remodellers such as ALC1/CHD1L, CHD2 or CHD7 could be required for Ku70/80 and the rest of the NHEJ machinery to associate with DSBs within chromatinized DNA 7–10. However, these mechanisms and the ability of Ku70/80 and DNA-PKcs to promote repair in the context of chromatin remain unclear.

Results
To determine whether Ku70/80 can bind to DNA when packaged within a nucleosome, we designed a nucleosome construct containing the minimum DNA length of 147 bp to encircle the histone octamer. We first purified Ku70/80 from insect cells to a high purity (Supplementary Fig. 1) and reconstituted 147 bp nucleosomes. Using electromobility shift assays (EMSAs) we were able to confirm the binding of Ku70/80 to the nucleosome, with a stable band present at a ratio of 1:4 (nucleosome: Ku70/80) (Supplementary Fig. 2A). We then prepared cryo-EM grids of this complex sample and following data collection and processing (Supplementary Fig. 3 and Table 1), we produced a map to a global resolution of 3.3 Å (Fig. 1B). We could confidently dock a nucleosome with the 147 bp Widom “601” positioning sequence and a single Ku70/80 molecule. The modelled Ku70/80 molecule is positioned such that the DNA from the nucleosome enters through the preformed ring of Ku70 and Ku80 encircling the duplex DNA. The structure of Ku70/80 when bound to the nucleosome is indistinguishable from previous structural studies of Ku70/80 in complex with short lengths of DNA as shown in (Supplementary Fig. 7A). Ku70/80 covers approximately 10 bp of DNA when bound in this structure, as calculated using PISA analysis 11 (Supplementary Fig. 8A). This model therefore confirms that Ku70/80 can bind nucleosomal DNA in the absence of DNA flanking the nucleosome.

Within our initial nucleosome: Ku70/80 map, there was weak density visible corresponding to a second Ku70/80 molecule bound on the opposite side of the nucleosome on the other exposed DNA end. We may be observing a mixture of Ku70/80 bound to either DNA end in our dataset, which is being averaged out. In an attempt to resolve the density for this second Ku70/80 molecule, we increased the length of the DNA to 153 bp. Following grid preparation of Ku70/80 with the 153 bp nucleosome, data collection and processing, we resolved a cryo-EM map to an overall resolution of 3.5 Å. Within this map, we could model two Ku70/80 molecules within the density present on both DNA ends (Fig. 1C). Interestingly, in the 147 bp nucleosome map, the N-terminus of H3 could be seen coming into close proximity with Ku70/80. Similarly, H3 appears to directly contact Ku70/80 when the DNA length is increased to 153 bp. This H3 interaction site on Ku80 is the same region that interacts with the Ligase IV BRCT domain in the SRC (Supplementary Fig. 9). In addition to H3, the C-terminal tail of H2A appears to contact Ku70/80 only when the DNA length is increased to 153 bp (Fig. 1C). Although interactions between the histone tails and Ku70/80 were observed, direct binding between peptides, both with and without different post-translational modifications, and Ku70/80 was not detected in assays conducted by EpiCypher. In this assay, 280 biotinylated histone peptides were tested to see if they interact with Ku70/80. Interactions between the histone tails observed and Ku70/80 were not detected, and only potential interactions with histone tails far from the site of interaction were observed (Supplementary Fig. 10). These interactions may therefore be weak and only present in the context of nucleosome-Ku70/80 binding. The small increase in DNA length (3 bp to each DNA end) causes Ku70/80 to rotate, indicating its ability to thread onto the DNA in a corkscrew-like manner to some extent. However, like that identified in the 147 bp Ku70/80 structure, the Ku70/80 molecules were also identical in structural orientation to those previously reported and covered ~10 bp of DNA 12 (Supplementary Figs. 7B and 8B).

To determine whether Ku70/80 can continue to translocate inwards towards the nucleosome core upon increasing the DNA length (distance of the break from the NCP), we increased the DNA length to 197 bp. In this cryo-EM map (solved to an overall lower resolution of 7.6 Å), we could visualise the increase in the DNA length and two Ku70/80 molecules binding at the ends of the DNA chain (Fig. 1D). The DNA from the nucleosome still enters through the preformed ring of Ku70 and Ku80 encircling the duplex DNA, covering ~12 bp of DNA (Supplementary Fig. 8C). The limited resolution in this map is likely due to the flexibility of the Ku70/80 molecules on the extended DNA. However, the Ku70/80 molecules are clearly positioned distant to the NCP at the DNA termini, indicating that the corkscrew-like threading of Ku70/80 seen when increasing the DNA length from 147 to 153 is limited, and that Ku70/80 is unable to translocate along the DNA chain progressively. We can also again recognise that the Ku70/80 molecules are similar in structure to those previously determined 12 (Supplementary Fig. 7C). From the three varied DNA length nucleosome constructs, it appears that Ku70/80 does not have a preference for linker length.

Comparing the models generated from the three different nucleosome DNA lengths, it must first be noted that the mode of Ku70/80 binding is identical in all three nucleosome constructs, with the DNA binding to the heterodimer as previously determined 12 (Supplementary Fig. 7A–C). It can also be seen that in the 147 and 153 bp DNA, binding of Ku70/80 causes the DNA to bend away from the NCP by approximately 25° to allow threading of Ku70/80 (Fig. 1E and Supplementary Fig. 11). This corroborates previous studies suggesting “peeling” of the DNA away from the NCP upon Ku70/80 binding 13. This may also be caused by unwrapping of the nucleosomal DNA that allows Ku70/80 to capture the nucleosomal DNA ends 14,15. However, when the DNA length was increased to 197 bp, the DNA followed its canonical path on the NCP. This indicates that the peeling of the DNA is caused by a steric hindrance, and once Ku70/80 captures the DNA ends, they need to be displaced from the histone octamer. However, once the DNA length is increased, the DNA ends are more readily accessible/flexible to Ku70/80 binding and do not require peeling away from the NCP.

In all our cryo-EM maps of Ku70/80 with nucleosomes, we do not observe multiple copies of Ku70/80 loading onto a single DNA end. It has been shown that multiple Ku70/80 molecules can load onto linear DNA in vitro; however, this is limited to ~1–2 molecules in cells 14–17. It has recently been shown that DNA-PKcs is responsible for preventing multiple loading of Ku70/80 on chromatin. Ku70/80 accumulation is further restricted by a neddylation/FBXL12-dependent process that actively removes loaded Ku70/80 molecules throughout the cell cycle and a CtIP/ATM-dependent mechanism that operates in S-phase 18. Likely, we do not see multiple Ku70/80 molecules loading in our cryo-EM data, as this is sterically inhibited by the relatively limited length of DNA used. Additionally, it could be considered that the ratio used in our cryo-EM experiments (1:4, nucleosome: Ku70/80) is not permissive, or the loading of multiple Ku70/80 molecules requires active chromatin remodelling.

Phosphorylation of the H2AX variant is a well-known early step in the response to DNA damage, which can be performed by the kinase activity of DNA-PKcs. It is known to primarily phosphorylate H2AX at serine 139. To assess whether the presence of H2AX in the nucleosome alters the binding activity of Ku70/80, we reconstituted the 153 bp nucleosome with H2AX replacing canonical H2A (Supplementary Fig. 12) and, similar to the H2A-containing nucleosomes, we performed binding and structural assays. As with canonical H2A, EMSA gels with H2AX containing nucleosomes confirmed the formation of a stable Ku70/80-nucleosome complex at a 1:4 (nucleosome: Ku70/80) ratio (Fig. 2A). Following cryo-EM data collection and data processing, two cryo-EM maps were produced, one corresponding to the 153 bp nucleosome bound to two molecules of Ku70/80 in a similar arrangement to the 153 bp nucleosome containing canonical H2A (Fig. 2B). However, the second map contained additional density corresponding to the Ku70 C-terminal linker and SAP domain (Fig. 2C) (to overall resolutions of 2.9 and 3.4 Å, respectively). The Ku70 C-terminal linker leads down from the core of Ku70/80 and appears to engage with the negatively charged (acidic) patch formed by histones H2AX-H2B (Fig. 2B). This acidic patch is a characteristic feature of the nucleosome surface and is a common site of attachment of various regulatory chromatin proteins 19. When compared to known H2A binders such as Dot1L, SAGADUB and COMPASS, a clear overlap can be observed between the linker of Ku70 and these protein complexes. However, it is not clear whether any specific residues overlap between the structures (Supplementary Fig. 13). The SAP domain itself binds on top of the nucleosomal DNA, with residues K596 and T577. It should be noted that we did previously observe some density corresponding to the Ku70 linker and SAP domain in the 153 bp H2A nucleosome datasets, highlighting that this density is not specific to H2AX (Supplementary Fig. 14). The density within the H2A nucleosome map is however, not as easily resolved as in the H2AX dataset, which may be due to data quality or stabilisation of the flexible tail of H2AX. We modelled the linker and SAP domain of Ku70 by docking a known structure of the SAP domain and tracing the linker density up to the core of Ku70. The density for this region of Ku70 is low resolution and was difficult to confidently model amino acid side chains. Notably, AlphaFold 3 20 was unable to accurately predict the position of the Ku70 SAP domain or linker region (Supplementary Figs. 15 and 16).

Similar to the nucleosome construct with 153 bp and canonical H2A (Fig. 1C), the H3 N-terminal and H2AX C-terminal density can be seen to lead towards Ku70/80. However, no additional density was observed that would indicate a specific interaction with Ku70/80 through these histone tails.

To determine the effect of the SAP domain of Ku70 binding to DNA, we also carried out EMSA studies comparing full-length Ku70/80 binding to DNA and Ku70/80-ΔSAP (see “Methods”) binding to DNA (Supplementary Fig. 17A). This EMSA gel shows that in the absence of the SAP domain, a higher band shift can be observed. This suggests that the SAP domain controls multiple loading of Ku70/80 onto DNA, and in its absence, multiple copies of Ku70/80 are able to bind. This is similar to what has recently been observed with the removal of the SAP domain 21. Furthermore, we also utilised AlphaFold 3 20, to analyse the SAP domain binding to a 20 bp DNA duplex. Interestingly, AlphaFold also predicts the binding of the SAP domain to DNA with K596 interacting directly with the DNA (Supplementary Fig. 17B), as we see in our cryo-EM structure (see further details below). It also shows that the overall SAP domain shows a positively charged patch which binds to the negatively charged DNA (Supplementary Fig. 17D). Furthermore, the Ku70 SAP domain in isolation interacts directly but weakly with a 400 bp DNA fragment in a manner that is stabilised specifically by residues K596 and T577 as evidenced by EMSA analysis (apparent KD in the 0.5 to 1 mM range). WT vs K596 (Fig. 2E) and WT vs T577 (Supplementary Fig. 17F), therefore showing the importance of these residues in this interaction. Additionally, EMSAs comparing WT SAP and K596A SAP binding to 153 bp free DNA and 153 bp nucleosome-bound DNA demonstrate that the SAP domain alone is capable of interacting with both DNA substrates (Fig. 2F). The SAP domain displays a clear preference for free DNA, as evidenced by the more pronounced mobility shift observed in the corresponding EMSA gels. Consistent with the results obtained using 400 bp free DNA, the K596A mutation markedly reduces the SAP domain’s binding affinity for both free and nucleosome-bound DNA. Collectively, these results indicate that the SAP domain can associate with both free and nucleosome-bound DNA, and that residue K596 is critical for this interaction.

To determine the importance of the interaction between the Ku70 linker and/or SAP domain with chromatin in vivo, we expressed in human U2OS cells these domains fused to GFP, in the absence of the rest of the Ku70 protein. The linker-SAP region of Ku70 (residues 532–609) exhibits a nuclear tropism, likely driven by the presence of the Ku70 Nuclear Localisation Signal (NLS) (Fig. 2H) 22, as well as a high affinity for chromatin with strong chromosome staining in mitotic cells. The linker alone (residues 532–554), which lacks part of the NLS, partially leaks into the cytoplasm and loses its strong binding to chromatin. Conversely, the SAP domain (residues 555–609), despite lacking most of the NLS and partially leaking into the cytoplasm, retains its high affinity for chromatin. To assess the role of the very C-terminal part of the NLS present in the SAP construct, a slightly extended version of the linker domain (linker+: residues 532–557) retaining the entire NLS was tested. Although this construct now localises mainly in the nucleus, it still displays no particular binding to chromatin, suggesting that within the linker-SAP region of Ku70, the SAP domain per se is sufficient to support this strong binding. To further characterise the SAP/chromatin interaction, we mutated the linker-SAP region of Ku70 on residues identified by cryo-EM as potentially interacting with nucleosomes (Fig. 2G). As mentioned above, these residues were difficult to confidently model due to low resolution, but were based on the most likely interactions as compared to previous proteins interacting with the acidic patch. However, in agreement with the above results, mutations on residues R543 and K553 (contacting the acidic patch) lying in the linker domain have no effect on chromatin binding. On the other hand, although the T577A mutation has no effect either, mutations on K596 (K596A and K596E) completely abolish the strong binding of the Linker-SAP domain to chromatin (Fig. 2I, J). These results suggest that the SAP domain of Ku70 contacts chromatin in vivo in a manner similar to the interaction identified by cryo-EM between Ku70/80 and nucleosome DNA. We therefore propose that the SAP domain is important for DNA binding, whether that be free DNA or packaged within nucleosomes, while the linker region mediates more nonspecific interactions that may vary depending on the DSB site.

Recent studies investigated the role of the Ku70-SAP domain. It has been shown that the Ku70-SAP domain is important in restricting Ku70/80 rotation and lateral movement on DNA, which is largely masked by DNA-PKcs 21. Using a mouse model, it was shown that deletion of the Ku70 SAP domain did not affect V(D)J recombination or animal development but did impair the animals’ and cells’ ability to repair exogenously induced DSBs 23. The SAP mutant cells were unable to retain Ligase IV and additional NHEJ factors on chromatin. Therefore, our structural and functional insights highlight the role and importance of the Ku70 SAP domain in Ku70/80 binding to DSBs in the cell.

It has been shown recently through measurements in living cells that Ku70/80 and DNA-PKcs arrive at the broken DNA ends simultaneously 24. Therefore, we next wanted to explore the recruitment of DNA-PKcs to form the DNA-PK holoenzyme complex. Following successful purification of DNA-PKcs from HeLa nuclear extracts (Supplementary Fig. 1), we collected cryo-EM data of Ku70/80 and DNA-PKcs together with a 153 bp nucleosome (containing H2AX) in a ratio of 1:4:2 (DNA: Ku70/80: DNA-PKcs, shown to work with linear DNA previously 2). Following processing of this dataset (Supplementary Fig. 18 and Table 2), we generated three distinct cryo-EM maps (Fig. 3). The first map (solved to a global 4.5 Å resolution) and corresponding model (Fig. 3A) show Ku70/80 bound to the nucleosome, with density corresponding to DNA-PKcs on the opposite side of Ku70/80 to form the DNA-PK holoenzyme monomer. In this structure, the position of the nucleosome relative to Ku70/80 has changed compared to our structures of Ku70/80 bound to nucleosomes in the absence of DNA-PKcs and is closer to Ku70 (Fig. 3A). In this map, the vWA domain of Ku80 is in a closed conformation, whilst the Ku70 vWA domain is slightly open, as seen previously for DNA-PK cryo-EM structures 2. The second map (solved to 4.4 Å resolution) also shows a DNA-PK monomer, but here the nucleosome is engaged further away from Ku70, in a similar orientation to Ku70/80 bound to a 153 bp nucleosome in the absence of DNA-PKcs. Ku70 is open to a similar degree as seen in model/map 1 and previous DNA-PK structures; however, in this structure, the Ku80 vWA domain is in an “open” conformation, which has not previously been identified without the addition of XLF or other accessory NHEJ protein factors 25 (Fig. 3B and Supplementary Fig. 22). It should be noted that Ku70/80 binds to the nucleosomal DNA in a similar manner in these structures when compared to Ku70/80 alone, with the DNA entering the central DNA binding pocket. Again, approximately 13 bp are within the Ku70/80 DNA binding pocket, whereas only ~8 bp are covered by DNA-PKcs, likely due to the short length of DNA (Supplementary Fig. 23). The nucleosome conformation also, as mentioned, differs between model 1 and model 2, with this movement coinciding with the opening of the vWA domain of Ku80 (Supplementary Fig. 7D, E). Furthermore, the observed interactions between the histone tails and Ku70/80 are no longer clearly visualised. Remarkably, we no longer observed density for the C-terminal linker and SAP domain of Ku70 within these DNA-PK nucleosome maps. We do, however, regularly observe density at the bottom of the circular cradle of DNA-PKcs, which we predict is the SAP domain of Ku70 (Supplementary Fig. 7G). Therefore, the SAP domain is able to relocate from the NCP to DNA-PKcs upon formation of DNA-PK.

Finally, the third map from this dataset shows a Ku80-mediated DNA-PK dimer (solved to 4.4 Å resolution) (Fig. 3C). This shows the same overall Ku80-mediated dimer structure previously identified 2, but now with a nucleosome on either side of Ku70/80 within this dimeric assembly. In this structure, Ku70 vWA displays the same open conformation as previously shown, with Ku80 adopting an intermediate open state when compared to the monomeric DNA-PK maps 2 (Supplementary Fig. 22). From direct comparisons with model 1 and model 2 DNA-PK monomer nucleosome structures, the DNA-PK dimer model appears more similar to model 2, where Ku70 and Ku80 are open. You can clearly see that in model 1, the NCP appears to have moved compared to model 2 and the DNA-PK dimer (Supplementary Fig. 24). Like with the monomeric DNA-PK models, density is no longer observed for the Ku70 C-terminal linker and SAP domain engaged with the NCP, however, unlike with the DNA-PK monomers we cannot observe any density on DNA-PKcs corresponding to the SAP domain. Notably, within this DNA-PK nucleosome dataset, we can identify an additional low-population conformation with density extending from the head domain of DNA-PKcs, near the kinase active site (Fig. 3D). Even though this density is low resolution, we can confidently dock into this a molecule of Ku70/80. The Ku70/80 molecule is positioned such that the bridge region of Ku70/80 is close to the kinase site of DNA-PKcs. This positioning of an additional Ku70/80 molecule has not been previously observed, and we hypothesise that this additional Ku70/80 may be primed for phosphorylation by DNA-PKcs.

After obtaining maps of DNA-PK bound to nucleosomes, we subsequently sought to activate DNA-PKcs by the addition of ATP (grids frozen after ~15 min and 1 h post-ATP), to determine if any structural changes are induced upon kinase activation when bound to nucleosome DNA. Surprisingly, following cryo-EM data collection and processing, we again were able to solve three maps identical to those identified when no ATP was added to the sample (Supplementary Fig. 25). This may be due to the fast turnover of ATP and the absence of further NHEJ proteins such as Ligase IV, XRCC4 and XLF, which are required for the DNA repair mechanism to proceed. To therefore “trap” DNA-PKcs, we added the non-hydrolysable ATP analogue AMP-PNP (see “Methods”) to DNA-PK with nucleosomes. Strikingly, we could observe large filaments on the micrographs (Fig. 4A), not seen with samples of only DNA-PK and nucleosome, or when ATP was added. Following cryo-EM data processing, we could observe extended repeating units within the 2D class averages (Fig. 4B), producing a final cryo-EM map to a global resolution of 5 Å (Fig. 4C). In this map, we were able to model the Ku80-mediated DNA-PK dimer bound to a nucleosome on either side (Fig. 3C), with an additional Ku70/80 molecule on either side of the nucleosome. We could observe that ~5 bp and 15 bp enter DNA-PKcs and Ku70/80, respectively, as shown in the previous structures, and ~17 bp in the additional Ku70/80 molecule (Supplementary Fig. 26). We can also observe extended map density for another DNA-PKcs molecule on either side to create an extended filament bridged by bound nucleosomes. We created a model of this extended filament of DNA-PK and nucleosomes based on our cryo-EM data. In this experimental map, we do not observe density for the additional Ku70/80 molecule close to the kinase site, as shown in Fig. 3. We do not observe clear density for AMP-PNP in the active site, which has been shown previously 26. This may be due to the relatively modest resolution of this map. The repeating unit of this filament is very similar to the DNA-PK dimer model shown in Fig. 3C. It is apparent, however, that the addition of AMP-PNP results in a striking structural stabilisation. We propose that the stabilised filament structure observed upon the addition of AMP-PNP reflects that this trapped state favours the formation of the Ku80-mediated LRC and ultimately should enhance DNA synapsis. This may explain how DNA-PK quickly couples synapsis with kinase activation, leading to more efficient DNA repair.

Attempts to solve structures of more complex NHEJ machineries in complex with nucleosomes (with the addition of Ligase IV, XRCC4 and XLF) proved unsuccessful, resulting in maps that represented only sub-assemblies of this machinery. For example, using the 153 bp nucleosome construct produced only maps of DNA-PK bound to nucleosomes. However, this is perhaps not surprising, as when we docked the XLF-mediated LRC onto our cryo-EM structures, we observed a steric clash with the stalk of XRCC4 and BRCT domains of Ligase IV with the nucleosome (Supplementary Fig. 27). We subsequently extended the DNA, but could still only visualise DNA-PK bound to nucleosomes. While it may be that further optimisation of this complex is necessary, we additionally hypothesise that a subsequent remodelling step of the nucleosome DNA is required before further NHEJ proteins can be engaged. It has been recently shown that the Artemis: DNA-PKcs nuclease complex does not act on mono-nucleosome substrates, highlighting the steric challenges posed by nucleosomes 27.

Notably, all the cryo-EM data presented here is generated without the use of chemical crosslinkers to stabilise specific structural states, which is unusual in the literature for either nucleosome or NHEJ structures, and we believe indicates the structures we observe have the potential to form in the cell (rather than trapping transient/biologically irrelevant states). From the data presented, we propose a model of DSB recognition by the NHEJ machinery in the context of nucleosomes (Fig. 5). Following initial DSB formation, Ku70/80 is the first protein to recognise the broken DNA ends, binding with high affinity even in the context of nucleosomes. The action of Ku70/80 binding results in the DNA partially “peeling-off” the histones. The position of the break with respect to the nucleosome dyad dictates the specific orientation of Ku70/80 and interactions with H3 and H2A(X) histones. Ku70/80 will always stay at the end of the DNA break and does not freely translocate inwards. DNA-PKcs is then subsequently recruited to form the DNA-PK holoenzyme complex. The binding of DNA-PKcs to nucleosome-bound Ku70/80 causes the vWA domain of Ku80 to open. Opening of the vWA domain of Ku80 has only previously been observed upon additional NHEJ proteins, such as XLF binding 1,3,25. Opening of the vWA domain without a known binding partner highlights the importance of DNA-PK binding to nucleosome DNA, potentially “priming” DNA-PK to interact with further NHEJ factors (however, our experiments have not been able to capture complexes formed with additional NHEJ factors). The Ku80-mediated DNA-PK dimer is then stabilised during ATP binding and/or hydrolysis. This dimer is presumably relatively short-lived in the cell, as the addition of non-hydrolysable ATP significantly stabilises this structural form. Remodellers are then likely to be required to remodel the DNA from the nucleosome to allow the recruitment of additional NHEJ proteins and ligation of the broken DNA ends.

In summary, we have captured how Ku70/80 and DNA-PK bind and interact with DSBs when packaged within nucleosomes, allowing us to propose a model for biological NHEJ DNA repair (Fig. 5). We have solved multiple cryo-EM structures tracking the initial DSB recognition and Ku70/80 binding, recruitment of DNA-PKcs and stabilisation of the Ku80-mediated DNA-PK dimer. Our findings reveal unique insights into this critical DNA repair pathway, which will have direct importance for the development of future therapeutics. Specifically understanding the complex mechanism of NHEJ with more physiologically relevant structures (in the context of chromatin) may reveal new unexplored drug pockets, the interplay with chromatin re-modellers in the early stage of repair and unique steps of the mechanism to target. Structural data plays a critical role in rational therapeutic design. Inhibiting NHEJ is beneficial during chemo- and radiotherapy in cancer treatments, and this information paves the way for developing more specific ways to target NHEJ and other repair pathways. Until now, the primary focus for targeting DNA-PKcs has been its ATP-binding site; however, inhibitors directed at this site often lead to significant side effects. The availability of new structural data reveals additional interaction surfaces, such as the interface between the Ku70 C-terminus and the DNA, even when packaged within the nucleosome, which may offer opportunities for developing more selective and less toxic therapeutic agents in the future.

Methods
Ku70/80 expression and purification
A pFASTBac-dual expression vector encoding full-length Ku70/80 and Ku70/80-ΔSAP (composed of Ku70 (residues 1–544) + Ku80 full length) with an N-terminal 6× His-tag on Ku80 were expressed in Sf9 insect cells. Following expression, Ku70/80 was purified by immobilisation onto Ni-NTA beads and anion exchange chromatography. All purification steps were carried out at 4°C. Cell pellets were resuspended in Lysis Buffer (20 mM Tris, 150 mM KCl, 850 mM NaCl, 5 mM β-mercaptoethanol (BME), 40 mM Imidazole, 1× protease inhibitor tablet, pH 8.0) and sonicated on ice. The resultant cell lysate was incubated, mixing, with MgCl2 (final concentration 10 mM) and 2 µL Benzonase (25 kU of stock) for 20 min and centrifuged at 18,000 × g for 40 min. The supernatant was incubated with Ni-NTA resin (Takara Bio) previously equilibrated with Lysis Buffer for 1 h, and Ku70/80 was eluted with Elution Buffer (20 mM Tris, 150 mM NaCl, 5 mM BME, 300 mM Imidazole, pH 8.0). The elution fractions were analysed on a 10% SDS-PAGE gel, and those containing Ku70/80 were pooled and dialysed against 2 L Buffer A (20 mM Tris, 50 mM NaCl, 50 mM KCl, 5 mM BME, pH 8.0) overnight at 4°C. The dialysed sample was applied to a 5 mL HiTrap Q column equilibrated in Buffer A, and Ku70/80 was eluted using a linear gradient of Buffer B from 0–40% (20 mM Tris, 150 mM NaCl, 850 mM KCl, 5 mM BME, pH 8.0). Fractions spanning the 280 nm absorbance peak were analysed on a 10% SDS-PAGE gel, the fractions containing Ku70/80 at a high purity were pooled, concentrated and buffer exchanged into cryo-EM Buffer (20 mM HEPES, 200 mM NaCl, 0.5 mM EDTA, 2 mM MgCl2, 5 mM DTT, pH 7.6) using a 50 kDa molecular weight cut-off (MWCO) centrifugal filler (Pierce) and flash-frozen in liquid nitrogen before being stored at -80°C for further use.

DNA-PKcs purification
Frozen HeLa Cell Nuclear Extracts were initially dialysed into 5 L Buffer A (20 mM HEPES, 100 mM NaCl, 0.5 mM EDTA, 2 mM MgCl2, 5 mM DTT, 0.2 mM PMSF, 10 complete protease inhibitor cocktail EDTA-free tablets, 10% (vol/vol) glycerol, pH 7.6). DNA-PKcs was isolated using several ion exchange columns: Q Sepharose, HiTrap Heparin, Mono-S and Mono-Q with a NaCl gradient of 0.1–1 M using Buffer B. 4–12% Bis-Tris gels (Invitrogen) were used to confirm the presence and purity of DNA-PKcs. Fractions containing DNA-PKcs were pooled, concentrated, and buffer exchanged into cryo-EM Buffer using a 10 kDa MWCO centrifugal filter (Amicon) and flash-frozen in liquid nitrogen before being stored at -80°C for further use.

Histone expression and purification
A vector encoding full-length H2A(X)/H2B/H3/H4 were transformed into Rosetta pLysS cells. A single colony was picked and grown in a 2xYT starter culture before being transferred into 1 L 2xYT media and incubated shaking at 37°C until the OD 600nm reached between 0.6 and 0.8. Protein expression was induced with 0.4 mM IPTG and incubated at 37°C for 4 h, and cells were harvested.
Cell pellets were resuspended in Histone Wash Buffer (50 mM Tris, 100 mM NaCl, 5 mM DTT, 1× protease inhibitor tablet, pH 7.5) and were sonicated on ice and centrifuged at 30,000 × g for 20 min at 4°C. The pellet was washed 2× with Histone T-wash Buffer (+2% Triton-100) and then 2× with Histone Wash Buffer before being resuspended in 0.75 mL DMSO and incubated, rolling for 30 min at room temperature. 7.5 mL of Unfolding Buffer (7 M Guanidine Hydrochloride, 20 mM Tris, 10 mM DTT, pH 7.5) was then added and incubated at room temperature, rolling, for 1 h. When the pellet was fully dissolved, Histone Purification Buffer A (7 M Urea, 50 mM Tris, 1 mM EDTA, pH 7.5) was added to bring the total volume to 30 mL before centrifugation. The histone solution was filtered (0.22 µm) and loaded onto a 5 mL HiTrap Q HP column; the flow through was collected and subsequently loaded onto a 5 mL HiTrap HP SP column. Histones were eluted by applying a linear gradient of Histone Purification Buffer B (+2 M NaCl) from 0–100%. Fractions spanning the 280 nm absorbance peak were analysed on a 15% SDS-PAGE gel. Fractions containing pure histones were pooled and dialysed against 2 L Buffer (5 mM DTT, 0.1% acetic acid, 1× protease inhibitor tablet) and freeze-dried in 2 mg aliquots and stored at -80°C until required.

Histone octamer assembly
Individually purified histones H2A(X), H2B, H3 and H4 were resuspended in an Unfolding Buffer (7 M Guanidine hydrochloride, 20 mM sodium acetate, 10 mM DTT, pH 5.2) and mixed in a ratio of 1:1:1:1 at a total protein concentration adjusted to 1 mg per histone in Unfolding Buffer. The histone mix was then dialysed 3× against 2 L Refolding Buffer (2 M NaCl, 10 mM Tris, 1 mM EDTA, 5 mM BME, pH 7.5) at 4°C, stirring for 2 h, 2 h and overnight. The dialysed sample was injected onto a Superose 6 Increase 10/300 equilibrated with Refolding Buffer. Fractions spanning the main 280 nm absorbance peak were analysed on a 20% SDS-PAGE gel. Fractions containing four histone bands were pooled, concentrated using a 10 kDa molecular weight cut-off centrifugal filter (Amicon) and stored at 4°C for further use.

DNA preparation and nucleosome assembly
147 and 197 bp. The 147 bp and 197 bp DNA were purchased as ssDNA oligos (IBA Lifesciences) and subsequently amplified by PCR using Taq polymerase (NEB), reaction cycles followed as per the manufacturer’s suggestion. The nucleosome DNA and primer sequences can be found in Tables 3 and 4, respectively.
Nucleosome assembly was carried out through the salt gradient dialysis method 6. The H2A/H2B dimer and H3.1/H4 tetramer at concentrations 1.3 µM and 0.65 µM, respectively, were obtained (EpiMark® Nucleosome Assembly Kit, NEB) and mixed with the appropriate DNA at a concentration of 0.65 µM in Assembly Buffer (20 mM Tris, 1 mM DTT, 1 mM EDTA, 0.01% Triton X-100, pH 7.5). The histone/DNA mixture was dialysed against Assembly Buffer (containing 2 M NaCl) for 2 h, followed by Assembly Buffer containing decreasing concentrations of NaCl at 1.5 M, 1 M, 0.6 M, 0.25 M for 2 h each, respectively and finally 0 M NaCl, overnight, at 4°C, stirring. Nucleosome assembly was confirmed through native PAGE gel analysis and concentrated to a final concentration of 5 µM and stored at 4°C for further use.
153 bp. DNA purification and nucleosome reconstitution followed previously established protocols 28,29. pUC19 plasmid containing 32× 153 bp 601 Widom DNA sequence 30 (Supplementary Table 3), was transformed into DH5α cells. A single colony was inoculated into 50 mL LB and grew for 4 h before being inoculated into 750 mL LB, shaking overnight at 37°C. Cells were harvested and pellets were resuspended in Solution I (25 mM Tris, 50 mM Glucose, 10 mM EDTA, pH 8.0), Solution II (200 mM NaOH, 1% SDS) and Solution III (3 M KOAc, 11% acetic acid) and incubated on ice for 15 min before centrifugation at 11,000 × g for 15 min at 4°C. The supernatants were filtered using Miracloth (Calbiochem) and 0.52× isopropanol was added. The mix was then incubated for 30 min and centrifuged at 11,000 × g for 30 min at 4°C. The resulting pellet was then incubated overnight at 37°C, rotating, with RNAse A (10 mg/mL). To purify the DNA, KCl was added to a final concentration of 2 M, and centrifuged at 11,000 × g for 35 min at 4°C. The supernatant was filtered (0.22 µm) and injected onto a Sepharose 6 column equilibrated with TES 2000 (50 mM Tris, 2 M KCl, 1 mM EDTA, pH 8.0). Fractions spanning the 260 nm absorbance peak were analysed on a 2% agarose gel stained using Ethidium Bromide. The fractions containing plasmid DNA were pooled, and 0.5× isopropanol was added, and centrifuged at 22,000 × g for 20 min at 4°C. The pellet was resuspended in TE 10, 0.1 (10 mM Tris, 0.1 mM EDTA, pH 8.0) and incubated at 50°C until the DNA was fully dissolved. The plasmid DNA was digested with EcoRV overnight at 37°C. To precipitate the 153 bp DNA fragment of interest, PEG 6000 (40%) with 4 M NaCl was added to a final concentration of 8% and 0.5 M NaCl, incubated on ice for 30 min and centrifuged at 11,000 × g for 30 min at 4°C. Samples of the pellet and supernatant were run on a 2% agarose gel to confirm the presence of the 153 bp DNA fragment. The pellet was resuspended in TE 10, 0.1 and stored at 4°C until further use.
153 bp DNA was added to a final concentration of 5 µM to varying ratios of the histone octamer, to find the optimal loading of the octamer onto DNA to minimise free DNA in the sample. Ratios 0.6:1.0 to 1.0:1:0 (histone octamer: DNA) were prepared. The assembled samples were placed into dialysis chambers as described in ref. 31 and subject to stepwise dialysis from the Reconstitution Buffer (1.4 M KCl, 10 mM Tris, 0.1 mM EDTA, pH 7.5) into the End Buffer (10 mM KCl, 10 mM Tris, 0.1 mM EDTA, pH 7.5) at 4°C, stirring. The dialysed samples were removed from the dialysis chambers and centrifuged at 20,000 × g for 10 min at 4°C. To confirm nucleosome assembly, the samples were run on a 5% acrylamide native gel, against a control of 153 bp free DNA. The nucleosome sample at an equimolar ratio was stored at 4°C and used in subsequent experiments.

EMSA studies of nucleosomes with Ku70/80 or Ku70/80-ΔSAP. For EMSA experiments, different nucleosome to Ku70/80 ratios were attempted to establish which ratio resulted in the best binding of Ku70/80. The nucleosome concentration was kept constant at 50 nM, and Ku70/80 was added in increasing ratios, after assembly samples were incubated on ice for 30 min and run on a 4–12% TBE gel (Invitrogen) for 45 min at 150 Volts with 1× TBE Running Buffer at 4°C. The best results were obtained when nucleosomes were mixed with Ku70/80 in a 1:4 ratio (nucleosome: Ku70/80). EMSA gels were stained using Sybr GOLD (Invitrogen) and imaged under UV light to visualise DNA.

Purification of the human Ku70 SAP domain in isolation
A gene fragment (GenScript) coding for the human Ku70 SAP domain was subcloned in frame between the SfoI—XhoI sites in pSF228 32,33 to generate pMM3046 (Ku70 SAP residues 539–609) for inducible expression in bacteria as a HIS14-SUMObd-Ku70 SAP fusion (the SUMObd moiety is from Brachypodium distachyon. In addition, constructs for producing mutant Ku70 SAP domains, pMM3047 (K596A) and pMM3047 (T577E), were similarly generated. All plasmids were verified by DNA sequencing and are available upon request.
The SUMObd-Ku70 SAP fusion plasmids were transformed into bacterial Rosetta 2 (DE3) pLysS cells (Novagen), which were grown in standard LB medium (2 L scale) supplemented with kanamycin (25 mg/mL) and chloramphenicol (34 mg/mL) at 37°C until OD 600nm reached 0.6. The temperature of the culture was then dropped to 15°C, and expression was induced by the addition of IPTG (0.25 mM final concentration) and further incubation for 16 h. Cells were collected by centrifugation, resuspended in 10 mL of PBS supplemented with 10 mM EDTA, and stored at -20°C. The cell paste was thawed and resuspended with one volume of 1.6 M NaCl, 40 mM Tris-HCl, pH 7.5, 2 mM EDTA, 2 mM DTT, 20 mM imidazole, 0.2% Triton X-100, 20% glycerol, supplemented with protease inhibitors (Pierce). The lysate was treated by sonication and clarified by centrifugation at 20,000 × g. The cleared lysate (~50 ml) was injected through a 5 mL HisTrap excel column (Cytiva) equilibrated with 0.8 M NaCl, 20 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM DTT, 10 mM imidazole, 10% glycerol, washed with 10 column volumes of the same buffer, and the fusion protein was eluted by increasing the imidazole concentration to 300 mM. The eluate was dialysed at 4°C for 16 h against 150 mM KCl, 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, 10% glycerol, supplemented with His14-SENP1 to cleave the SUMObd moiety. The dialysate was injected through a 5 mL HisTrap excel column equilibrated with 150 mM KCl, 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, and 10% glycerol to recover the untagged Ku70 domain in the flow through. Yields for a 2 L culture were 40 mL at 10 μM, which could readily be concentrated to around 200 μM using 3 kDa MWCO ultrafiltration devices (PALL). Proteins in 150 mM KCl, 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, 10% glycerol were snap frozen in liquid N2 and stored at -80°C.

EMSA with the human KU70 SAP domain in isolation
Binding reactions (10 mL) containing the indicated concentrations of Ku70 SAP protein and 19.3 nM of a 400 bp DNA fragment (generated by PCR using primers 5′-GAGTTTTATCGCTTCCATGAC and 5′-ACTTGACTCATGATTTCTTACC, with PhiX174 DNA (NEB) as the template) were prepared in 105 mM KCl, 3 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 0.1 mM DTT, and 4% glycerol. Reactions were incubated for 15 min at room temperature before fractionation on a 0.8% agarose gel (10 cm) in 0.5× TBE buffer, run at 50 Volts for 2 h. Following electrophoresis, gels were stained with GelRed (Biotium) and the fluorescent signal was detected with a ChemiDoc MP imaging system (BioRad).
For assays containing the 153 bp DNA substrate or 153 bp nucleosome, binding reactions were prepared as above, using 50 nM of either free DNA or nucleosome substrate in 46 mM KCl, 3 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, and 3% glycerol. Reactions were incubated and resolved as described above on a 1.5% agarose gel run at 80 V for 120 min at 4°C. Gels were stained with GelRed and imaged as described.

Preparation of cryo-EM samples
Ku70/80 bound to 147, 153 H2A(X) and 197 bp nucleosome: 3.5 μM of the appropriate nucleosome was mixed with 14 μM of Ku70/80, respectively (1:4 ratio) and kept on ice for 30 min. Prior to freezing grids, 3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) was added to a final concentration of 8 mM to help circumvent the preferred orientation of particles within vitreous ice. 2.5 μl was then applied to a glow-discharged (30 mA, 30 s) holey carbon grid (Quantifoil 1.2/1.3, 300 mesh) and plunge-frozen into liquid ethane using the FEI Vitrobot system (Thermo Fisher Scientific) with the chamber kept at 4°C and 95% humidity.
DNA-PK bound to 153 bp H2AX nucleosome: 3 μM of the appropriate nucleosome was mixed with 12 μM of Ku70/80 and 6 μM DNA-PKcs, respectively (1:4:2 ratio) and kept on ice for 30 min. CHAPSO was added to a final concentration of 8 mM, and 2.5 μl was then applied to a glow-discharged (30 mA, 30 s) holey carbon grid (Quantifoil 1.2/1.3, 300 mesh) and plunge-frozen into liquid ethane using the FEI Vitrobot system (Thermo Fisher Scientific) with the chamber kept at 4°C and 95% humidity.
DNA-PK bound to 153 bp H2AX nucleosome with AMP-PNP: The complex was assembled as described above, and AMP-PNP was then added to a final concentration of 1 mM and incubated on ice for 1 h before the addition of CHAPSO and grid freezing.

Cryo-EM data collection: Cryo-EM data for the Ku70/80-147 bp nucleosome complex were collected at the Department of Biochemistry, University of Cambridge, on a Krios electron microscope operated at 300 kV using a K3 detector (Gatan Inc.) with EPU data acquisition software (Thermo Fisher Scientific). The Ku70/80-197 bp nucleosome complex was collected at the Department of Materials Science, University of Cambridge, on a Krios electron microscope operated at 300 kV using a Falcon III detector with EPU data acquisition software (Thermo Fisher Scientific).
Cryo-EM data of all other data presented were collected at The Midlands Regional Cryo-EM facility, University of Leicester, on a Krios electron microscope operated at 300 kV using a K3 detector (Gatan Inc.) with EPU data acquisition software (Thermo Fisher Scientific). All data collection parameters can be found in (Supplementary Tables 1 and 2).

Data processing: For the Ku70/80-147 and -197 bp nucleosome complexes, data were processed using Warp 34 and CryoSPARC 35. In short, motion correction, CTF correction and particle picking were performed using Warp, and particles were extracted in BoxNet2Mask using a box size of 380 and 370 for the 147 and 197 bp nucleosome complexes, respectively. All other data presented were solely processed using CryoSPARC.
The extracted particles were processed using different workflows in cryoSPARC (Supplementary Figs. 3 and 18). In summary, particles were selected and optimised through iterative rounds of 2D classification, ab-initio reconstruction and heterogeneous refinement to separate the Ku- or DNA-PK-nucleosome complexes from free nucleosomes and proteins. The best maps were then further refined using homogenous refinement and finally non-uniform refinement 36. Local refinements were also completed to improve the density in localised areas. For the obtained final reconstructions, the overall resolution (Å) (displayed in Supplementary Tables 1 and 2) was calculated by Fourier shell correlation at 0.143 cut-off.

Building and refinement of cryo-EM models: For the Ku70/80 bound to 147, 153 (H2A) and 197 bp nucleosome, the model of Ku70/80 (PDB: 1JEY) and for the Ku70/80 bound to 153 bp H2AX nucleosome, the model of Ku70/80 (PDB: 7ZT6) were used as initial templates and rigidly fit into the cryo-EM density maps in UCSF Chimera 37. For DNA-PK maps, the model of DNA-PK (PDB: 8BH3) was used, with chains other than DNA-PKcs and Ku70/80 manually deleted.
The nucleosome in the 147 bp model (PDB: 7TEEN) and 197 bp (PDB: 6TEM), and for all other models nucleosome (PDB: 6JXD) was used as an initial model. The DNA was manually mutated to the correct sequence, and the DNA extensions were built using the “build” ideal DNA tool in WinCoot 38 and manually fit into the density. The model of the SAP domain (from PDB: 1JEQ) was rigidly fitted in UCSF Chimera, and the linker region was manually built in WinCoot. Once all initial models were generated, Namdinator 39 was used to improve the map to model fit. Following this, each model went through several rounds of real-space refinement in Phenix 40 and improvement in WinCoot until ready for PDB deposition.

Cell lines, cell culture and cell engineering: U2OS cells (human osteosarcoma cell line from ECACC, Salisbury, UK) were grown in 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.
Production of lentiviral particles in HEK-293T cells and transduction of U2OS cells were performed as previously described 41.
U2OS cells expressing the different GFP-tagged Ku70 Linker-SAP constructs were seeded in 35-mm glass-bottom culture dishes (MatTek) 48 h prior to imaging with an Olympus IX73 fluorescence microscope equipped with an Olympus LCAch N 20×/0.40 PhC and a UPlanFLN 40×/0.75 Ph2 objective lenses. Images were taken with an Olympus DP26 camera.

Expression vectors and DNA manipulations: A Ku70 lentiviral expression vector was generated by PCR amplification of human Ku70 cDNA with Ku70-Kpn2-F and Ku70-Bcu-R primers and subsequent insertion between Kpn2I and BcuI restriction sites of the previously described pLV3 vector 25.
Expression vectors for full-length mutant forms of Ku70 were obtained by PCR mutagenesis with the corresponding Ku70-mut-F and Ku70-mut-R oligonucleotides as mutated inner primers and HF-Ku70-Kpn2-F and HF-Ku70-Bcu-R as outer primers, followed by Hot-Fusion insertion of the two overlapping fragments between the Kpn2I and BcuI restriction sites of pLV3 42.
The expression vector for GFP-tagged Ku70 constructs was obtained by replacing the FLAG-Ku70 cDNA from the previously described pLV3-GFP-FLAG-Ku70 vector 43 by a linker cassette (pre-annealed Kpn2-AX-Mlu-F and Kpn2-AX-Mlu-R oligonucleotides) between the Kpn2I and MluI sites, resulting in the pLV3-GFP plasmid. Expression vectors for GFP-tagged Linker, Linker+ or SAP domains were obtained by PCR amplification of the corresponding fragments using full-length Ku70 expression vector as a template with HF-GFP-Ku70-LS-F/HF-Ku70-R554-R, HF-GFP-Ku70-LS-F/HF-Ku70-V557-R or HF-GFP-Ku70-P555-S-F/HF-Ku70-Bcu-R pairs of primers for Linker, Linker+, or SAP domains, respectively. The resulting PCR fragments were inserted by Hot-Fusion between the Kpn2I and BcuI restriction sites of pLV3-GFP.
The expression vectors for mutated Ku70 Linker-SAP constructs were obtained in a similar manner by PCR amplification of the corresponding fragments using full-length Ku70 expression vectors as templates and HF-GFP-Ku70-LS-F and HF-Ku70-Bcu-R as outer primers.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript by Hall et al., determines the structural basis of the early stages of NHEJ in the context of chromatin. This is\naccomplished by determining a series of cryo-EM structures that show how the Ku70/80 heterodimer initially engages the\nterminal ends of a nucleosome during DSB recognition, and how the Ku70/80/DNA-PKc complex assembles on the “broken”\nnucleosomal DNA ends. These structural insights are timely and substantially increase our understanding for how DNA\nrepair is initiated in chromatin. The work will undoubtedly be well-received in the DNA repair field. However, I do have\nseveral comments/concerns related to the quality of the structural models, the structural interpretations, and the cellular\nassays probing the Ku70 SAP-Linker domain function that the authors should consider and address prior to publication in\nNature Communications.\n1. The maps are generally not of high enough quality to model the side chain residues within Ku70/80 and Ku70/80/DNA-\nPKc, and in some cases the NHEJ machinery is modeled in places where there is clearly minimal interpretable cryo-EM\ndensity. I strongly suggest the authors go back through these models and prune side chains and remove regions Ku70/80\nand Ku70/80/DNA-PKc that do not have interpretable cryo-EM density. This will also likely improve the elevated clash\nscores for many of these structures.\n2. The results section of the manuscript is quite difficult to get through. Part of the issue is the sheer number of different\nstructures the authors walk through, but in most cases this is done in a single sentence or two. For example, the description\nof the Ku70/80 structure bound to the 197bp WT nucleosome simply states Ku70/80 binds to the end of the linker DNA.\nDoes it thread through the Ku70/80 heterodimer? How does it compare to the mode of Ku70/80 binding to the 147 bp and\n153 bp WT nucleosomes? Providing more than a surface level description of many of these structures would likely improve\nthe readability of the manuscript and allow the readers to better track the major take-home message from each of the\nstructures.\n3. The authors show quite nicely through complex formation assays that Ku70/80 binds to nucleosomes with varying lengths\nof linker DNA and to nucleosomes containing histone variants associated with the DNA damage response (e.g., H2Ax).\nHowever, the lack of quantitative binding affinities reported within the manuscript makes it very difficult to interpret whether\nKu70/80 has any preference for nucleosomes with a specific linker DNA length or nucleosomes with variants. This also\nmakes interpreting the Ku70/80 cryo-EM data difficult, especially given that differences in Ku70/80 binding mode were\nobserved for the different nucleosome substrates (e.g., the SAP domain binding to the acidic patch in H2Ax nucleosomes\nbut not WT nucleosomes). Additional quantitative binding assays would significantly strengthen the manuscript and allow for\nmore robust interpretation of the structural findings. Alternatively, the authors should provide at least some commentary on\nwhether they believe Ku70/80 has some preference for linker DNA of different lengths\nand/or the histone H2A variants.\n4. The putative interaction between the Ku70/80 complex and histone tails in the 147bp WT nucleosome, 153bp WT\nnucleosome, and 153bp H2Ax nucleosome structures is an intriguing finding. However, it’s unclear whether these\ninteractions are important for Ku70/80 binding to the nucleosome, or simply the result of Ku70/80 binding to the nucleosomal\nDNA ends placing it in proximity to the H3 and C-terminal H2A tail. In addition, it was difficult to track why these interactions\nwere seen in some of the structures but not others? Without higher resolution structural information where the interaction can\nbe seen in detail, or supporting biochemical assays that show this interaction is important for Ku70/80 complex binding to\nnucleosomes, the authors should use caution when discussing the histone tail interactions throughout the manuscript.\n5. The observation that entry/exit site DNA is partially unwrapped when bound by Ku70/80 in the 147bp and 153bp\nnucleosomes could also be explained by spontaneous unwrapping of the nucleosomal DNA that allows Ku70/80 to capture\nthe nucleosomal DNA ends (see PMID: 1525856), rather than Ku70/80 physically “peeling” the nucleosomal DNA off the\nhistone octamer after binding. This alternative interpretation should at least be mentioned in the manuscript as well as the\ndiscussion.\n6. The authors note that that they did not observe two Ku70/80 heterodimers bound to the same DNA end in all of the\ndifferent nucleosomes structures they determined with varying in linker DNA length (Lines 131-142). The cryo-EM\nprocessing workflow in Extended Fig. 2 lacks any of the intermediate maps generated during processing, which makes it\nimpossible to evaluate a statement such as this. While I appreciate the attempts to consolidate as much of the cryo-EM\nprocessing into single figures, Extended Fig. 2 (and to some extent Extended Fig. 6) are overly complicated making it\ndifficult to track the processing workflow for each individual dataset.\n7. Can the authors comment on why the Ku70 SAP-linker only interacts with the acidic patch and nucleosomal DNA in the\nH2Ax nucleosome complex but not the WT nucleosome complex? This intriguing finding was given minimal explanation.\n8. The experiments testing the importance of the SAP domain and linker region of Ku70 in U2OS cells are problematic.\nSome of these issues and points requiring clarification are below:\n• Why did the authors choose to study the Ku70 SAP and linker domain without the rest of the Ku70 protein?\n• It appears that the SAP domain and/or linker mutants were never tested for their ability to bind nucleosomes in vitro prior to\nmoving into the cellular experiments. This makes interpreting these experiments extremely difficult, as it’s unclear whether\nthese mutants actually disrupt the SAP-linker domain interaction with the nucleosome that was observed in the structures.\n• Were these cells treated with DNA damaging agents to generate DSBs to recruit Ku70/80 to chromatin? If not, is it possible\nthat some of the mutants that show null results are because the SAP-linker interface is important for DSB recognition in\nchromatin rather than just general chromatin binding?\n• Did the authors attempt to quantify any of these experiments?\n• I was unable to find the methods section for this set of experiments, which should be addressed.\n9. The structures of Ku70/80/DNA-PKc (non-activated) bound to the H2Ax nucleosome are quite remarkable. The authors\ndid an excellent job walking through the different conformational states related to Ku80 vWa opening/closing. However, they\nfail to describe how the Ku70/80/DNA-PKc complex bound to the H2Ax nucleosome differs from the earlier described\nKu70/80 complex bound to H2Ax nucleosome. Does the overall conformation of the nucleosome change between these\nstructures? Does Ku70/80 bind to the nucleosome in a similar manner in these structures regardless of the presence of\nDNA-PKc? What happens to the SAP-linker domain interaction with the acidic patch and the nucleosomal DNA? My own\ninitial comparisons of these structures suggest quite a few interesting differences that were either overlooked or ignored.\nThis type of structural analysis if quite important given that the novelty of this work is rooted in understanding how the NHEJ\nmachinery functions within a chromatin context (on a nucleosome substrate).\n10. A similar comparison of the Ku70/80/DNA-PKc bound to the H2Ax nucleosome (non-activated states) and the\nKu70/80/DNA-PKc bound to the H2Ax nucleosome (activated states) should be made (see preceding comment).\n11. The section of the discussion on drug development is quite superficial. What unique insights were gleaned from these\nstructures that will enhance the development of therapeutics?\nMinor Comments\n1. Several of the structural models provided with the manuscript have components that vary in chain ID from model to model,\nwhich made it quite difficult to compare/contrast the structures (e.g., compare Ku70/80 bound to 153bp H2Ax nucleosome\nand Ku70/80/DNA-PKc bound to 153bp H2Ax nucleosome-model 1). The consensus used in the field is that Chain A/E\n(Histone H3), Chain B/F (Histone H4), Chain C/G (Histone H2A), Chain D/H (Histone H2B), Chain I/J (Nucleosomal DNA). I\nwould suggest the authors renumber the chains within the models for uniformity between their own structures, but also for\nuniformity with virtually all other nucleosome structures in the PDB.\n2. The manuscript contains numerous grammatical errors and labeling errors. Some, but not all, are highlighted below.\n3. Lines 91-92: A figure showing a structural comparison of Ku70/80 engaged with the nucleosome and the previously\ndetermined structures of Ku70/80 bound to DNA would be useful.\n4. Extended Data Fig. 2: Particle numbers for 147bp DNA have commas in the wrong place.\n5. Lines 99-100: This sentence lacks clarity.\n6. Lines 140-141: I believe “permissive” should be “not permissive”.\n7. Lines 167-171: This section should be edited for clarity. The resolution of this 153bp nucleosome bound by two Ku70/80\nshould be given in the sentence it was described in rather than the sentence after.\n8. Lines 179-180: This sentence lacks clarity.\n9. Line 200: I believe “K543” should be “R543” based on Figure 2b.\n10. Line 256-258: This statement requires a citation.\n11. Lines 358-360: I would caution the authors in describing this as “unwinding the DNA.”", "reviewer_2": "The premise of this study is both interesting and promising. DNA breaks in eukaryotes occur within chromatin, and thus DNA\ndouble-strand breaks (DSBs) are surrounded by nucleosomes. However, no studies to date have shown how the repair\ninitiators Ku70/80 and DNA-PKcs interact with nucleosomes.\nIn this manuscript, the authors report structures of Ku70/80 bound to the end(s) of 147 bp (Widom 601 sequence), 153 bp\nand 197 bp DNA on a nucleosome. In these constructs, however, the nucleosome is positioned centrally with both DNA ends being symmetrically equal in length. These configurations do not reflect the typical scenario in which only one end is\naccessible and free for binding by Ku70/80, or by both Ku70/80 and DNA-PKcs. The authors determined cryoEM structures\nof these three nucleosomes bound by Ku70/80. As expected, 147 bp and 153 bp nucleosomes do not present enough free\nDNA for Ku70/80 and DNA-PKcs to bind, so DNA ends are peeled off from the histones. In the case of the 147 bp\nnucleosome, only one end is peeled off, resulting in partial binding by Ku70/80.\nIn addition, the authors analyzed potential effects of the histone variant H2AX, which replaces H2A on damaged DNA, on\nKu70/80 binding, as well as the influence of DNA-PKcs and ATP on the combined binding of Ku70/80 and DNA-PKcs to\nthese nucleosomes. These findings raise more questions than answers (see comments below). Due to the lack of\nstructural knowledge regarding DSB repair in the context of nucleosomes, the results presented in this manuscript are novel and\nwarrant consideration for publication. However, several issues related to DNA substrate design and data interpretation\nshould be addressed first. A scientist should recognize the limitations of each experiment and interpret the results based on\nexperimental data and established scientific knowledge.\n1. The three nucleosome DNAs used in this study as substrates for DSB repair are unrealistic. First, in a cellular context, two\nDNA breaks surrounding a nucleosome would likely lead to the loss of the nucleosome rather than its repair. Second, it is\nhighly improbable that a DSB can occur so close to or even within a nucleosome, such as the 147 bp one, because a\nhistone octamer would either slide off or move inward from the broken end, even if the DSB occurs immediately adjacent to a\nnucleosome, regardless of the presence of nucleosome remodelers. Third, the 601 Widom sequence binds tightly to a\nhistone octamer and is not prone to sliding in either direction. This is evident from the structures of the 153 bp nucleosome-\nKu70/80 complex, where only a limited number of DNA base pairs were “peeled” off at each end, and neither end results in\noptimal binding by Ku70/80.\n2. The authors observed that one end of the DNA in the 147-bp nucleosome is associated with Ku70/80 when a 4-fold\nexcess of Ku70/80 is mixed with the nucleosome. Is the DNA end bound by Ku70/80 unique, or is it a mixture of both DNA\nends? Did the histone core translocate along the DNA?\n3. How many base pairs are associated with Ku70/80 in the 147-bp nucleosome? Typically, a minimum of 14 bp are covered\nby one Ku70/80 molecule (PMID: 11493912). In the case of the 147 bp Widom nucleosome, approximately 140 bps are\ninvolved in histone binding. Peeling off any stretch of DNA to allow Ku70/80 to bind would be energetically costly. Ku70/80\nbinds free DNA ends with a dissociation constant of ~ 1 nM; however, the Kd of Ku70/80 when associated with nucleosomes\nappears to range from 50 to 100 nM, indicating it is 50-100 times weaker. Could such weak binding occur in vivo?\n4. The authors asserted multiple times that Ku70/80 is the first to arrive at broken DNA ends. What evidence do they have to\nsupport this statement? Recent measurements in living cells indicate that Ku70/80 and DNA-PKcs arrive at broken DNA\nends simultaneously (PMID: 39578493). Furthermore, as quoted by the authors in reference #23, DNA-Pkcs limits excessive\nloading of Ku70/80. If Ku70/80 arrives before DNA-PKcs, how does DNA-PKcs prevent the loading of multiple Ku70/80\nonto a DNA end?\n5. Human Ku70/80 typically slides along free DNA and does not remain at the DNA end in the absence of DNA-PKcs.\nTherefore, the observation of Ku70/80 remaining at the DNA ends on the 197-bp nucleosome is unusual. Is there steric\nhindrance preventing Ku70/80 from sliding toward the nucleosome? For studying Ku70/80 and DNA-PKcs binding, an ideal\nnucleosome may consist of 175 bp, leaving one free DNA end of 30 bp.\n6. The cell data reporting SAP binding to chromatin is promising. However, it does not differentiate whether SAP binds to the\nacidic patch of the histone or the DNA, nor does it specify whether it interacts with H2A or H2AX. As a result, the implications\nof SAP binding to the acidic patch in the 153-bp nucleosome containing H2AX remain uncertain. Given the spatial\nconstraint, it is possible that SAP docking on the histone acidic patch, which is common to both H2A and H2AX, could be\ncoincidental.\n7. As the acidic patch exists in both H2A and H2AX, have the authors measured the binding affinity of the SAP domain of\nKu70 with H2A/H2B compared to H2AX/H2B? Additionally, how does SAP binding to the acidic patch compare to that of\nknown binder of H2A, such as Dot1L, SAGA DUB or COMPASS?\n8. In the presence of DNA-PKcs, how many base pairs of the 153 bp nucleosome are bound by DNA-PKcs? A\nsuperimposition of the structure of DNA-PK bound to free DNA with that of the 153-bp nucleosome would be helpful. Only\nwhen 15 bp are bound to DNA-PKcs is the kinase activated. Have the authors measured the DNA-PKcs kinase activity in\nthe presence of 147- or 153-nucleosome?\n9. Given that the DNA end is blunt and open, did the authors observe specific interactions between DNA-PKcs and the DNA\nend, such as the DEB (DNA end blocking) helix of DNA-PKcs between the DNA strands? DEB binding to the DNA end may\nslow down but not completely inhibit DNA-PKcs kinase activity.\n10. Given the extremely short DNA length available for DNA-PKcs binding and the lack of evidence supporting DNA-PKcs\nactivation, the conclusions regarding the roles of ATP and AMP-PNP in promoting DNA-PK dimerization and the “activated\nstate” of DNA-PKcs are speculative, if not entirely unfounded.", "reviewer_3": "The authors present cryo-electron microscopy structures of the human Ku70/80 heterodimer and DNA-PK holoenzyme\nbound to nucleosomes with varying DNA lengths, to investigate how non-homologous end joining machinery recognizes\nDNA double-strand breaks in chromatin. They show that Ku70/80 binding can unwrap nucleosomal DNA and find that the\nKu70 SAP domain binds nucleosomal DNA in the Ku complex. Further interactions are found at the histone acidic patch and\ninvolve the linker region between the Ku80 core domain and the SAP domain. Additionally, they reveal a conformational\nchange in Ku80 upon DNA-PK binding and demonstrate that non-hydrolysable ATP promotes DNA end synapsis.\nAltogether, these findings advane our understanding of the binding of Ku to chromatinized DNA and provide structural\ninsights into DSB repair by NHEJ. All in all, the paper is well written and the structural analysis generally solid. The paper\ncovers structural biology up to some cell based assays. The area of research is of interest to both basic and biomedical\nsciences. In general, I believe the manuscript is of interest to the readers of Nature Communications. However, there are\nsome parts where I believe the evidence needs to be solidified before I can recommend publication.\nMajor points:\n1) Line 171/ Line 179-180: Is the appearance of the extra density and the proposed stabilisation of the flexible tail specific to\nH2AX? This is somewhat surprising since the acidic patch is also present in canonical histones. Are there any differences\nbetween H2AX and H2A at contact points between Ku and the nucleosome? Or can altered DNA unwrapping account for\nthe observed difference, and if so, would that be specific to H2AX? To validate the differences in binding, perhaps you could\nperform a competition experiment to see whether H2AX nucleosomes are bound better than H2A nucleosomes?\n2) What is the origin of the specific model shown in Figure 2E? The authors do not really show detailed density for that part\nand even state that they could not confidently identify specific histones contacts solely from cryo-EM maps. How can they\nconfidently model the linker? Does AlphaFold predict this interaction? I am not yet convinced on the basis of the provided\ndata for the physiological relevance of the SAP and linker nucleosome binding. Since this is a main part and conceptual\nadvance in this manuscript, I suggest that the authors better characterise this interaction in vitro, e.g. using a construct that\ncontains only linker and SAP domains and demonstrate nucleosome and acidic patch binding and work out any\ndifferences between H2AX and H2A nucleosomes.\nMinor and editorial points:\nLine 53: Perhaps define DNA-PK as DNA-dependent protein kinase first before abbreviating it.\nLine 161: Please indicate at which position DNA-PKcs phosphorylates H2AX.\nFigure 2 A and C : These panels overlap with gel labels (“Nucleosomes”) covered by Figure 2C.\nFigure 2 and general: Consider standardising the use of capital vs. lowercase letters.\nExtended Figure 2 : The local refinement depictions appear somewhat blurry (also in Extended Figure 5). In general, the\nprocessing scheme images are not very sharp. Perhaps this is due to the downsampled review files, but if not, please\nconsider improving their resolution and contrast."}, "author_response": "Manuscript number: NCOMMS-25-32754-T\nCryo-EM structures of NHEJ assemblies bound to nucleosomes\nResponse to Reviews\nThank you for arranging the review of our manuscript for Nature Communications. We thank\nall the reviewers for their careful consideration of the manuscript and were pleased to read that\nthey agreed the results increase our understanding of the NHEJ machinery and its interaction\nwith chromatin. We have now addressed the reviewer’s comments in our significantly revised\nsubmission and have detailed our responses below. We have included additional data analysis\nof all the structures with multiple new supplementary figures, redeposited the improved PDBs\nand added additional EMSA studies to analyse the Ku70 SAP domain importance.\nWe look forward to your response to our changes.\nYour Sincerely,\nAmanda Chaplin and co-authors\nREVIEWER COMMENTS\nReviewer #1:\nThis manuscript by Hall et al., determines the structural basis of the early stages of NHEJ in\nthe context of chromatin. This is accomplished by determining a series of cryo-EM structures\nthat show how the Ku70/80 heterodimer initially engages the terminal ends of a nucleosome\nduring DSB recognition, and how the Ku70/80/DNA-PKc complex assembles on the\n“broken” nucleosomal DNA ends. These structural insights are timely and substantially\nincrease our understanding for how DNA repair is initiated in chromatin. The work will\nundoubtedly be well-received in the DNA repair field. However, I do have several\ncomments/concerns related to the quality of the structural models, the structural\ninterpretations, and the cellular assays probing the Ku70 SAP-Linker domain function that\nthe authors should consider and address prior to publication in Nature Communications.\nWe thank the reviewer for mentioning that this work is timely and substantially increases our\nunderstanding for how DNA repair is initiated in chromatin. We appreciate the comments\nand will respond in detail below.\n1. The maps are generally not of high enough quality to model the side chain residues within\nKu70/80 and Ku70/80/DNA-PKc, and in some cases the NHEJ machinery is modelled in\nplaces where there is clearly minimal interpretable cryo-EM density. I strongly suggest the\nauthors go back through these models and prune side chains and remove regions Ku70/80\nand Ku70/80/DNA-PKc that do not have interpretable cryo-EM density. This will also likely\nimprove the elevated clash scores for many of these structures.\nWe appreciate the reviewers’ comment, although the cryo-EM density is clear for modelling\nthe individual proteins and DNA, we agree that in some areas of density for the side chain\nresidues is limited. We have now gone back through the data and improve where possible the\nmodelling by removing side chains which have no clear density. We have also modified the\nManuscript number: NCOMMS-25-32754-T\nCryo-EM structures of NHEJ assemblies bound to nucleosomes\nchain IDs to be consistent as mentioned by another reviewer. Hopefully this will enable\neasier comparisons between the structures.\n2. The results section of the manuscript is quite difficult to get through. Part of the issue is the\nsheer number of different structures the authors walk through, but in most cases this is done\nin a single sentence or two. For example, the description of the Ku70/80 structure bound to\nthe 197bp WT nucleosome simply states Ku70/80 binds to the end of the linker DNA. Does it\nthread through the Ku70/80 heterodimer? How does it compare to the mode of Ku70/80\nbinding to the 147 bp and 153 bp WT nucleosomes? Providing more than a surface level\ndescription of many of these structures would likely improve the readability of the\nmanuscript and allow the readers to better track the major take-home message from each of\nthe structures.\nWe apologise if the results section was too concise, due to the large number of structures we\ninitially felt a concise description of each would be clear and focused. However, we have\nnow expanded the results section and included greater detail, including information\nmentioned above for the 197bp structure. We have also added an additional supplemental\nfigure (Supplementary Fig. 7) showing how Ku70/80 with linear DNA (PDB: 1JEY)1\ncompares to Ku70/80 once bound to the nucleosomal DNA. We feel this broadens and\nexpands our description of the structures presented.\n3. The authors show quite nicely through complex formation assays that Ku70/80 binds to\nnucleosomes with varying lengths of linker DNA and to nucleosomes containing histone\nvariants associated with the DNA damage response (e.g., H2Ax). However, the lack of\nquantitative binding affinities reported within the manuscript makes it very difficult to\ninterpret whether Ku70/80 has any preference for nucleosomes with a specific linker DNA\nlength or nucleosomes with variants. This also makes interpreting the Ku70/80 cryo-EM data\ndifficult, especially given that differences in Ku70/80 binding mode were observed for the\ndifferent nucleosome substrates (e.g., the SAP domain binding to the acidic patch in H2Ax\nnucleosomes but not WT nucleosomes). Additional quantitative binding assays would\nsignificantly strengthen the manuscript and allow for more robust interpretation of the\nstructural findings. Alternatively, the authors should provide at least some commentary on\nwhether they believe Ku70/80 has some preference for linker DNA of different lengths\nand/or the histone H2A variants.\nWe thank the reviewer for this comment and agree quantitative affinities would be ideal.\nHowever, we should stress that we have been trying to obtain these over the last few years,\nand it appears to be more difficult than what one would expect. ITC is very difficult as it is\nnot possible to reconstitute nucleosomes at a high enough concentration. BLI has been tried\nand unfortunately was not successful. Ku70/80 was immobilised on BLI sensors via the His-\ntag on the protein, with the nucleosome present in the wells. Loading onto the sensors was\nsuccessful, however no association or dissociation was detected with nucleosomes. We\ntherefore tried our usual Y-shaped DNA as a positive control (known to have a nM affinity)\nand still limited association and dissociation was detected with a lot of background signal\n(DNA binding to reference, unloaded sensor). We have also tried biotinylated Ku70/80 and\nimmobilised onto streptavidin sensors (less background binding), however we still observed\nno association or dissociation. We now believe that having Ku immobilised is causing steric\nhindrance of the central DNA binding channel and not allowing binding. We have included\nthese results below to demonstrate everything we have tried in Figure 1 - below.\nManuscript number: NCOMMS-25-32754-T\nCryo-EM structures of NHEJ assemblies bound to nucleosomes\nWe agree that we should have made it clear in the manuscript that Ku70/80 does not have an\nobserved preference for DNA sequence or length or whether it is in the presence of H2A or\nH2AX. Although we report density for the Ku70-SAP domain in the H2AX structure, upon\nanalysis in our nucleosome data containing H2A, we do still observe some density for the\nSAP domain. We have now shown this in an additional supplemental figure (Supplementary\nFig. 14). This density was however more difficult to resolve than that presented for the\nH2AX dataset, but this is now described in more detail within the text. We have also now\nincluded an additional EMSA to show how when the SAP domain is removed from full-\nlength Ku protein that an increased band shift can be observed, again demonstrating the\nimportance of the SAP domain for DNA binding (Supplementary Fig. 17). Expression and\npurification of the SAP domain in isolation was also carried out, however it was not shown to\nbind to DNA with a high affinity and therefore was difficult to analyse the effect of the point\nmutation (Figure 2 - below).\nFigure 1: Biolayer Interferometry (BLI) and Flow induced dispersion analysis (FIDA)\nexperiments. A) Ni-NTA biosensor + His-tagged Ku70/80. Loading of Ni-NTA with His-Ku 200-25nM\n(+ reference “unloaded” sensor) for 600 seconds. Association into “analyte”, 153 bp H2AX nucleosome\n100nM for 300 seconds. No association or dissociation. B) Linear free DNA (Y-shape, walker) as a\npositive control for Ku binding. No association of dissociation between Ku70/80 and nucleosome DNA\nor linear DNA. Perhaps Ku70/80 being immobilised causes steric hinderance of the Ku70/80 DNA\nbinding channel. C) Biotinylated Ku70/80 and immobilised onto streptavidin biosensors. Ku70/80\nloading onto streptavidin sensors 500nM → 31.25nM. Analyte (y-shape DNA – Walker et al.,2001)\nconcentration 200nM. No association or dissociation between Ku70/80 and linear DNA. D-E) FIDA-\nBio experiments. D) Ku70/80 full-length protein. E) Ku70/80 truncated SAP. F) 153bp nucleosome. G)\nKu70/80 truncated SAP with 153 bp H2AX nucleosome at a ratio of 1:4 (5:20 µM). Averaging\ntechnique, not separation technique. Having an excess of Ku is masking any visualisation of a potential\ncomplex. However, to stay consistent with the EMSA and cryo-EM results it was assembled complex\nat 1:4 ratio.\nManuscript number: NCOMMS-25-32754-T\nCryo-EM structures of NHEJ assemblies bound to nucleosomes\nFigure 2: EMSA gels of Ku70 SAP domain. A) WT Ku70 SAP domain binding to DNA compared to\nK596 SAP mutant binding to 30 bp DNA. B) WT Ku70 SAP domain binding to DNA compared to\nK596 SAP mutant binding to 153 bp DNA. C) WT Ku70 SAP domain binding to DNA compared to\nK596 SAP mutant binding to 153 bp DNA with higher ratios and lower DNA concentration.\n4. The putative interaction between the Ku70/80 complex and histone tails in the 147bp WT\nnucleosome, 153bp WT nucleosome, and 153bp H2Ax nucleosome structures is an intriguing\nfinding. However, it’s unclear whether these interactions are important for Ku70/80 binding\nto the nucleosome, or simply the result of Ku70/80 binding to the nucleosomal DNA ends\nplacing it in proximity to the H3 and C-terminal H2A tail. In addition, it was difficult to track\nwhy these interactions were seen in some of the structures but not others? Without higher\nresolution structural information where the interaction can be seen in detail, or supporting\nbiochemical assays that show this interaction is important for Ku70/80 complex binding to\nnucleosomes, the authors should use caution when discussing the histone tail interactions\nthroughout the manuscript.\nWe again do thank the reviewer for this comment and recognise the limitations of these\nobserved interactions. We should mention that we have explored direct interactions between\nthe histone tails and Ku70/80 using the company EpiCypher. They explored interactions\nbetween 280 biotinylated histone peptides with and without post-translational modifications\nand their interactions with Ku70/80. However, they were unable to find a direct interaction\nbetween the histone tails we observe as interacting with Ku70/80. These interactions may\ntherefore only be present within the context of the nucleosomes. We have now added this\ninformation into the manuscript and as the reviewer mentions discussed these possible\ninteractions with caution (Supplementary Fig. 10).\n5. The observation that entry/exit site DNA is partially unwrapped when bound by Ku70/80\nin the 147bp and 153bp nucleosomes could also be explained by spontaneous unwrapping of\nthe nucleosomal DNA that allows Ku70/80 to capture the nucleosomal DNA ends (see\nPMID: 1525856), rather than Ku70/80 physically “peeling” the nucleosomal DNA off the\nhistone octamer after binding. This alternative interpretation should at least be mentioned in\nthe manuscript as well as the discussion.\nThis is a very nice alternative that we had not considered and have now included this\npossibility within the manuscript.\n6. The authors note that that they did not observe two Ku70/80 heterodimers bound to the\nsame DNA end in all of the different nucleosomes structures they determined with varying in\nlinker DNA length (Lines 131-142). The cryo-EM processing workflow in Extended Fig. 2\nlacks any of the intermediate maps generated during processing, which makes it impossible\nto evaluate a statement such as this. While I appreciate the attempts to consolidate as much of\nthe cryo-EM processing into single figures, Extended Fig. 2 (and to some extent Extended\nManuscript number: NCOMMS-25-32754-T\nCryo-EM structures of NHEJ assemblies bound to nucleosomes\nFig. 6) are overly complicated making it difficult to track the processing workflow for each\nindividual dataset.\nWe appreciate what the reviewer is saying, however, we must say these workflows did take a\nsignificant amount of time, in order to condense what would have been 9 additional figures.\nHowever, we do agree that they do not include intermediate maps and so we have now\nincluded these in additional figures (Supplementary Figs. 4, 5, 19 and 20).\n7. Can the authors comment on why the Ku70 SAP-linker only interacts with the acidic patch\nand nucleosomal DNA in the H2Ax nucleosome complex but not the WT nucleosome\ncomplex? This intriguing finding was given minimal explanation.\nWe apologise for the minimal explanation, as mentioned above we believe the SAP and\nlinker can bind when H2A is present but seems to appear stabilised and easily resolved with\nH2AX. We have now added additional description and detail about this including a figure\n(Supplementary Fig. 14) showing its presence in the H2A dataset.\n8. The experiments testing the importance of the SAP domain and linker region of Ku70 in\nU2OS cells are problematic. Some of these issues and points requiring clarification are\nbelow:\n• Why did the authors choose to study the Ku70 SAP and linker domain without the rest of\nthe Ku70 protein?\nWithout DNA-breaking treatment of cells, full-length (FL) Ku heterodimer exhibits a nuclear\nlocalization and is mostly extractable from chromatin, except a small RNAse-sensitive\nfraction 2, with no peculiar mitotic chromatin labelling. So, we chose to limit the analysis to\nthe Ku70 (Linker)-SAP region since its intrinsic chromatin tropism allowed exploration of\nmutations effects.\n• It appears that the SAP domain and/or linker mutants were never tested for their ability to\nbind nucleosomes in vitro prior to moving into the cellular experiments. This makes\ninterpreting these experiments extremely difficult, as it’s unclear whether these mutants\nactually disrupt the SAP-linker domain interaction with the nucleosome that was observed in\nthe structures.\nWe agree that in vitro nucleosomes binding experiments with WT and mutant linker-SAP-\nlinker would help the interpretation of the cellular data. As mentioned above we have now\nincluded an additional EMSA gel with and without the SAP domain, showing how the SAP\ndomain can control Ku70/80 loading (Supplementary Fig. 17). We have also attempted to\npurify the SAP domain and point mutation separately, and although the protein purified well,\nwe could not observe any binding to DNA using EMSAs (Figure 2 - above). Therefore, it\nwas not possible to carry out any further in vitro studies.\n• Were these cells treated with DNA damaging agents to generate DSBs to recruit Ku70/80 to\nchromatin? If not, is it possible that some of the mutants that show null results are because\nthe SAP-linker interface is important for DSB recognition in chromatin rather than just\ngeneral chromatin binding?\nThe cells were not treated with DNA breaking agents, so that the mitotic labelling we show is\nintrinsic and distinct from DSB recognition. We agree that some positions of the linker-SAP-\nlinker domain the mutation of which do not impact on intrinsic binding to chromatin may\ncontribute to full length Ku binding to DSB.\n• Did the authors attempt to quantify any of these experiments?\nManuscript number: NCOMMS-25-32754-T\nCryo-EM structures of NHEJ assemblies bound to nucleosomes\nWe did not attempt quantification since the effect of mutations were sharp, with either no\neffect (with the T577A SAP mutant, each mitotic chromatin was labelled as WT SAP\ncontrol) or full effect (no mitotic chromatin was labelled with either K596A or K596E SAP\nmutant).\n• I was unable to find the methods section for this set of experiments, which should be\naddressed.\nWe apologize for this inadvertent omission. Here is the corresponding method paragraph that\nwill be added to the Methods section (Cell engineering and imaging).\n“U2OS cells expressing the different GFP-tagged Ku70 Linker-SAP constructs were seeded\nin 35-mm glass-bottom culture dishes (MatTek) 48h prior to imaging with an Olympus IX73\nfluorescence microscope equipped with an Olympus LCAch N 20X/0.40 PhC objective lens.\nImages were taken with an Olympus DP26 camera.”\n9. The structures of Ku70/80/DNA-PKc (non-activated) bound to the H2Ax nucleosome are\nquite remarkable. The authors did an excellent job walking through the different\nconformational states related to Ku80 vWa opening/closing. However, they fail to describe\nhow the Ku70/80/DNA-PKc complex bound to the H2Ax nucleosome differs from the earlier\ndescribed Ku70/80 complex bound to H2Ax nucleosome. Does the overall conformation of\nthe nucleosome change between these structures? Does Ku70/80 bind to the nucleosome in a\nsimilar manner in these structures regardless of the presence of DNA-PKc? What happens to\nthe SAP-linker domain interaction with the acidic patch and the nucleosomal DNA? My own\ninitial comparisons of these structures suggest quite a few interesting differences that were\neither overlooked or ignored. This type of structural analysis if quite important given that the\nnovelty of this work is rooted in understanding how the NHEJ machinery functions within a\nchromatin context (on a nucleosome substrate).\nWe thank the reviewer for this comment and agree that a more detailed comparison is\nnecessary. We have now added additional text within the manuscript, highlighted in red and\nan additional figure Supplementary Fig. 7. The reviewer is right there are a few differences\nthat should have been highlighted, particularly the movement of the Ku70-SAP domain, that\nwe have now included.\n10. A similar comparison of the Ku70/80/DNA-PKc bound to the H2Ax nucleosome (non-\nactivated states) and the Ku70/80/DNA-PKc bound to the H2Ax nucleosome (activated\nstates) should be made (see preceding comment).\nWe again thank the reviewer and have now added additional textual comparisons and added\nan additional Supplementary Fig. 24.\n11. The section of the discussion on drug development is quite superficial. What unique\ninsights were gleaned from these structures that will enhance the development of\ntherapeutics? We have now added some more specific text on this.\nMinor\nComments\n1. Several of the structural models provided with the manuscript have components that vary\nin chain ID from model to model, which made it quite difficult to compare/contrast the\nManuscript number: NCOMMS-25-32754-T\nCryo-EM structures of NHEJ assemblies bound to nucleosomes\nstructures (e.g., compare Ku70/80 bound to 153bp H2Ax nucleosome and Ku70/80/DNA-\nPKc bound to 153bp H2Ax nucleosome-model 1). The consensus used in the field is that\nChain A/E (Histone H3), Chain B/F (Histone H4), Chain C/G (Histone H2A), Chain D/H\n(Histone H2B), Chain I/J (Nucleosomal DNA). I would suggest the authors renumber the\nchains within the models for uniformity between their own structures, but also for\nuniformity with virtually all other nucleosome structures in the PDB.\nWe have now gone through the models and changed the chain IDs so they are now consistent\nand make for easier comparison.\n2. The manuscript contains numerous grammatical errors and labeling errors. Some, but not\nall, are highlighted below.\n3. Lines 91-92: A figure showing a structural comparison of Ku70/80 engaged with the\nnucleosome and the previously determined structures of Ku70/80 bound to DNA would be\nuseful. We have now included this in Supplementary Fig. 7 and added additional text.\n4. Extended Data Fig. 2: Particle numbers for 147bp DNA have commas in the wrong place.\nThank you, this has now been changed.\n5. Lines 99-100: This sentence lacks clarity. This sentence has now been improved for\nclarity.\n6. Lines 140-141: I believe “permissive” should be “not permissive”. We have now changed\nthis.\n7. Lines 167-171: This section should be edited for clarity. The resolution of this 153bp\nnucleosome bound by two Ku70/80 should be given in the sentence it was described in rather\nthan the sentence after. This has now been changed.\n8. Lines 179-180: This sentence lacks clarity. This has now been deleted and reworded.\n9. Line 200: I believe “K543” should be “R543” based on Figure 2b. This has now been\nchanged.\n10. Line 256-258: This statement requires a citation. This has now been added.\n11. Lines 358-360: I would caution the authors in describing this as “unwinding the DNA.”\nThis has now been changed to remodel.\nReviewer #2\nThe premise of this study is both interesting and promising. DNA breaks in eukaryotes occur\nwithin chromatin, and thus DNA double-strand breaks (DSBs) are surrounded by\nnucleosomes. However, no studies to date have shown how the repair initiators Ku70/80 and\nDNA-PKcs interact with nucleosomes.\nWe thank the reviewer for noting that this study is both interesting and promising.\nManuscript number: NCOMMS-25-32754-T\nCryo-EM structures of NHEJ assemblies bound to nucleosomes\nIn this manuscript, the authors report structures of Ku70/80 bound to the end(s) of 147 bp\n(Widom 601 sequence), 153 bp and 197 bp DNA on a nucleosome. In these constructs,\nhowever, the nucleosome is positioned centrally with both DNA ends being symmetrically\nequal in length. These configurations do not reflect the typical scenario in which only one\nend is accessible and free for binding by Ku70/80, or by both Ku70/80 and DNA-PKcs. The\nauthors determined cryoEM structures of these three nucleosomes bound by Ku70/80. As\nexpected, 147 bp and 153 bp nucleosomes do not present enough free DNA for Ku70/80 and\nDNA-PKcs to bind, so DNA ends are peeled off from the histones. In the case of the 147 bp\nnucleosome, only one end is peeled off, resulting in partial binding by Ku70/80.\nIn addition, the authors analyzed potential effects of the histone variant H2AX, which\nreplaces H2A on damaged DNA, on Ku70/80 binding, as well as the influence of DNA-PKcs\nand ATP on the combined binding of Ku70/80 and DNA-PKcs to these nucleosomes. These\nfindings raise more questions than answers (see comments below). Due to the lack of\nstructural knowledge regarding DSB repair in the context of nucleosomes, the results\npresented in this manuscript are novel and warrant consideration for publication.\nAgain, we thank the reviewer for noting that the results presented in this manuscript are novel\nand warrant consideration for publication.\nHowever, several issues related to DNA substrate design and data interpretation should be\naddressed first. A scientist should recognize the limitations of each experiment and interpret\nthe results based on experimental data and established scientific knowledge.\n1. The three nucleosome DNAs used in this study as substrates for DSB repair are unrealistic.\nFirst, in a cellular context, two DNA breaks surrounding a nucleosome would likely lead to\nthe loss of the nucleosome rather than its repair. Second, it is highly improbable that a DSB\ncan occur so close to or even within a nucleosome, such as the 147 bp one, because a histone\noctamer would either slide off or move inward from the broken end, even if the DSB occurs\nimmediately adjacent to a nucleosome, regardless of the presence of nucleosome remodelers.\nThird, the 601 Widom sequence binds tightly to a histone octamer and is not prone to sliding\nin either direction. This is evident from the structures of the 153 bp nucleosome-Ku70/80\ncomplex, where only a limited number of DNA base pairs were “peeled” off at each end, and\nneither end results in optimal binding by Ku70/80.\nWe agree with the reviewer that the mono-nucleosomes used in this study do not represent\nthe situation in the cell. Rather, those structures represent our progress in moving the field\ntowards this eventual goal. To date, most of the structural work carried out on proteins\nbinding to nucleosomes has been in the context of mono-nucleosomes 3, 4. This is due to the\nhigh suitability of mono-nucleosomes for structural biology approaches compared to large\nnucleosome arrays and the significant advance in understanding of the molecular mechanisms\nthat are afforded by these structures. We believe using a nucleosome array will only result in\nvisualising Ku/DNA-PKcs bound to the DNA end as we show.\nSecondly, the 147 bp construct, we agree is close/within a nucleosome, however this was\nused initially based on the publication 5. This is why we increased the DNA length to 153 bp\nand eventually 197bp. The problem we faced when carrying out structural work, is that the\nlonger the DNA length the greater the flexibility due to the distance of Ku70/80 and DNA-\nPKcs from the NCP as shown in the 197 bp structure, which is significantly lower resolution.\nWe also believe that using a 147 bp nucleosome is not problematic and is a possible scenario.\nParticularly since it has been well documented that the histone octamer slides on the DNA\nand prefers to sit flush with the DNA ends 6.\nManuscript number: NCOMMS-25-32754-T\nCryo-EM structures of NHEJ assemblies bound to nucleosomes\n2. The authors observed that one end of the DNA in the 147-bp nucleosome is associated\nwith Ku70/80 when a 4-fold excess of Ku70/80 is mixed with the nucleosome. Is the DNA\nend bound by Ku70/80 unique, or is it a mixture of both DNA ends? Did the histone core\ntranslocate along the DNA?\nAs shown in Supplementary Fig. 6a, and copied below, within the 2D classes we can see\ndensity corresponding to Ku70/80 (shown with red circles) on either side of the nucleosome.\nTherefore Ku70/80 is able to bind to both ends of the 147 bp nucleosome, and perhaps we are\naveraging a mixture of it binding to both ends when creating the ab initio/3D models. This is\ndifficult to separate/distinguish in cryo-EM, but we have now added additional text to\nrecognise this. We do not observe any translocation of the histone core along the DNA, as\nmentioned above the 601 Widom sequence binds very tightly.\n3. How many base pairs are associated with Ku70/80 in the 147-bp nucleosome? Typically, a\nminimum of 14 bp are covered by one Ku70/80 molecule (PMID: 11493912). In the case of\nthe 147 bp Widom nucleosome, approximately 140 bps are involved in histone binding.\nPeeling off any stretch of DNA to allow Ku70/80 to bind would be energetically costly.\nKu70/80 binds free DNA ends with a dissociation constant of ~ 1 nM; however, the Kd of\nKu70/80 when associated with nucleosomes appears to range from 50 to 100 nM, indicating\nit is 50-100 times weaker. Could such weak binding occur in vivo?\nWe thank the reviewer and agree a more detailed analysis of how Ku70/80 binds to DNA\nwould be useful. Analysis of the DNA bases covered by Ku70/80 in the Ku70/80 +\nnucleosome structures have now been shown in Supplementary Fig. 8\nThe EMSA data we show is not enough to allow conclusion about the affinity of Ku70/80 in\nour system, however other techniques such as ITC, FIDA, BLI have not been possible due to\nvarious reasons as mentioned above and shown in Figure 1- above. Ku70/80 is a very\nabundant protein in the nucleus therefore if there are few other free DNA ends in the cell then\na nucleosomal end can be expected to be bound very well.\n4. The authors asserted multiple times that Ku70/80 is the first to arrive at broken DNA ends.\nWhat evidence do they have to support this statement? Recent measurements in living cells\nindicate that Ku70/80 and DNA-PKcs arrive at broken DNA ends simultaneously (PMID:\n39578493). Furthermore, as quoted by the authors in reference #23, DNA-Pkcs limits\nexcessive loading of Ku70/80. If Ku70/80 arrives before DNA-PKcs, how does DNA-PKcs\nprevent the loading of multiple Ku70/80 onto a DNA end?\nWe thank the reviewer for this comment, the traditional understanding of the NHEJ\nmechanism was that Ku70/80 would arrive first. However, we agree that recent\nmeasurements indicate the simultaneous binding with DNA-PKcs. We have now edited the\nManuscript number: NCOMMS-25-32754-T\nCryo-EM structures of NHEJ assemblies bound to nucleosomes\ntext, to remove the statements that Ku70/80 binds first and appreciate the DNA-PK formation\nmust occur as the reviewer mentions and referenced the paper above.\n5. Human Ku70/80 typically slides along free DNA and does not remain at the DNA end in\nthe absence of DNA-PKcs. Therefore, the observation of Ku70/80 remaining at the DNA\nends on the 197-bp nucleosome is unusual. Is there steric hindrance preventing Ku70/80 from\nsliding toward the nucleosome? For studying Ku70/80 and DNA-PKcs binding, an ideal\nnucleosome may consist of 175 bp, leaving one free DNA end of 30 bp.\nWe do not observe any significant sliding; however, the resolution of this cryo-EM map is\nlow, which we associate with flexibility, however it may indeed be due to Ku70/80\nmovement. We have now added some text to discuss this. We do not envisage the benefit of\nusing further nucleosome DNA substrates for this publication as we are focusing on the\ninitial recognition and synapsis and not ligation. However, we will consider using different\nDNA substrates for the subsequent NHEJ mechanism steps.\n6. The cell data reporting SAP binding to chromatin is promising. However, it does not\ndifferentiate whether SAP binds to the acidic patch of the histone or the DNA, nor does it\nspecify whether it interacts with H2A or H2AX. As a result, the implications of SAP binding\nto the acidic patch in the 153-bp nucleosome containing H2AX remain uncertain. Given the\nspatial constraint, it is possible that SAP docking on the histone acidic patch, which is\ncommon to both H2A and H2AX, could be coincidental.\nWe thank the reviewer for this comment. We should make it clear that the linker of Ku70 is\nwhat interacts with the acidic patch and that the SAP domain binds to the DNA. We have\nnow adjusted the text to make this clear. The cell data confirms that it is the SAP domain\nwhich is important for binding and not the linker. When looking back at previous cryo-EM\ndata, we do see some additional density for the SAP domain in other cryo-EM maps, however\nwe were never able to resolve this. Therefore, H2AX may or may not be important for\nstabilisation of Ku70 and this is now discussed further and more openly in the manuscript\nwith an additional figure (Supplementary Fig. 14).\n7. As the acidic patch exists in both H2A and H2AX, have the authors measured the binding\naffinity of the SAP domain of Ku70 with H2A/H2B compared to H2AX/H2B? Additionally,\nhow does SAP binding to the acidic patch compare to that of known binder of H2A, such as\nDot1L, SAGA DUB or COMPASS?\nWe thank the reviewer for this idea, although after careful consideration, we believe this\nexperiment would not give us any additional data. This is because the SAP domain binds to\nthe DNA, therefore without the DNA present we don’t believe the C-terminus of Ku70 would\nbe stabilised on only histones. This has been shown by our inclusion of data from EpiCypher\n(Supplementary Fig. 10). We have now made it clear in the text that the interaction between\nthe linker and the histone octamer is not the important interaction site, which we also discuss\nin the previous comment. We thank the reviewer for suggesting we compare the C-terminus\nof Ku70 interacting with the acidic patch with known binders of H2A. We have now added\nan additional Figure (Supplementary Fig. 13) and text to discuss the similarities and\ndifferences.\n8. In the presence of DNA-PKcs, how many base pairs of the 153 bp nucleosome are bound\nby DNA-PKcs? A superimposition of the structure of DNA-PK bound to free DNA with that\nof the 153-bp nucleosome would be helpful. Only when 15 bp are bound to DNA-PKcs is the\nManuscript number: NCOMMS-25-32754-T\nCryo-EM structures of NHEJ assemblies bound to nucleosomes\nkinase activated. Have the authors measured the DNA-PKcs kinase activity in the presence of\n147- or 153-nucleosome?\nWe have now added more comparison figures (Supplementary Figs. 7 and 24) and shown\nhow many DNA bps are covered by Ku70/80 and DNA-PKcs (Supplementary Figs. 8, 23\nand 26). We have carried out phosphorylation gels in the presence of 153 bp nucleosomes\nand can see that the kinase activity is not active. However, for the purpose of this study to\nlook at synapsis we believe this is not important. For future studies looking at transitions\nfrom long-range to short-range complexes we will extend the DNA length and check the\nability to carry out DNA-PKcs kinase activity.\n9. Given that the DNA end is blunt and open, did the authors observe specific interactions\nbetween DNA-PKcs and the DNA end, such as the DEB (DNA end blocking) helix of DNA-PKcs between the DNA strands? DEB binding to the DNA end may slow down but not\ncompletely inhibit DNA-PKcs kinase activity.\nThis is an interesting point; however, we do not observe the DEB helix in our data.\nGenerally, we only observe this when LX4 is engaged.\n10. Given the extremely short DNA length available for DNA-PKcs binding and the lack of\nevidence supporting DNA-PKcs activation, the conclusions regarding the roles of ATP and\nAMP-PNP in promoting DNA-PK dimerization and the “activated state” of DNA-PKcs are\nspeculative, if not entirely unfounded.\nWe completely agree with the reviewer and have now carefully formulated these conclusions\nin the manuscript.\nReviewer #3:\nThe authors present cryo-electron microscopy structures of the human Ku70/80 heterodimer\nand DNA-PK holoenzyme bound to nucleosomes with varying DNA lengths, to investigate\nhow non-homologous end joining machinery recognizes DNA double-strand breaks in\nchromatin. They show that Ku70/80 binding can unwrap nucleosomal DNA and find that the\nKu70 SAP domain binds nucleosomal DNA in the Ku complex. Further interactions are\nfound at the histone acidic patch and involve the linker region between the Ku80 core domain\nand the SAP domain. Additionally, they reveal a conformational change in Ku80 upon DNA-\nPK binding and demonstrate that non-hydrolysable ATP promotes DNA end synapsis.\nAltogether, these findings advance our understanding of the binding of Ku to chromatinized\nDNA and provide structural insights into DSB repair by NHEJ. All in all, the paper is well\nwritten and the structural analysis generally solid. The paper covers structural biology up to\nsome cell based assays. The area of research is of interest to both basic and biomedical\nsciences. In general, I believe the manuscript is of interest to the readers of Nature\nCommunications. However, there are some parts where I believe the evidence needs to be\nsolidified before I can recommend publication.\nWe thank the reviewer for these comments that the data advances our understanding of\nbinding Ku to chromatin, and that the structural analysis is solid.\nManuscript number: NCOMMS-25-32754-T\nCryo-EM structures of NHEJ assemblies bound to nucleosomes\nMajor points:\n1) Line 171/ Line 179-180: Is the appearance of the extra density and the proposed\nstabilisation of the flexible tail specific to H2AX? This is somewhat surprising since the\nacidic patch is also present in canonical histones. Are there any differences between H2AX\nand H2A at contact points between Ku and the nucleosome? Or can altered DNA unwrapping\naccount for the observed difference, and if so, would that be specific to H2AX? To validate\nthe differences in binding, perhaps you could perform a competition experiment to see\nwhether H2AX nucleosomes are bound better than H2A nucleosomes?\nWe thank the reviewer for this comment, and as mentioned above upon further analysis of\nour data we do observe some SAP density in the H2A structures, showing that the SAP is still\nable to bind. We do observe improved density with H2AX, which may be due to either the\nquality of the data or stabilisation through the extended tail. We have now added further text\nand Supplementary Fig. 14 to show this. In terms of biochemical analysis, as described\nabove we have found this aspect of the research very challenging with the nucleosomes due\nto various reasons mentioned above and shown in Figure 1- above. We are also unable to\ncarry out experiments of only H2A/H2B and the SAP domain because this requires DNA\nbinding within the context of the nucleosome. We have however added an additional EMSA\ngel (Supplementary Fig. 17) showing how the SAP domain regulates Ku loading to DNA.\n2) What is the origin of the specific model shown in Figure 2E? The authors do not really\nshow detailed density for that part and even state that they could not confidently identify\nspecific histones contacts solely from cryo-EM maps. How can they confidently model the\nlinker? Does AlphaFold predict this interaction? I am not yet convinced on the basis of the\nprovided data for the physiological relevance of the SAP and linker nucleosome binding.\nSince this is a main part and conceptual advance in this manuscript, I suggest that the authors\nbauthors\nbetter characterise this interaction in vitro, e.g. using a construct that contains only linker and\nSAP domains and demonstrate nucleosome and acidic patch binding and work out any\ndifferences between H2AX and H2A nucleosomes.\nThe model shown in Figure 2E was from docking the known SAP domain PDB into density\nfor the SAP and tracing density corresponding to the linker from the SAP domain up to\nKu70. We agree with the reviewer, although we could trace density for the linker we cannot\nbe confident of specific residues in exact locations, due to limited resolution for amino acid\nside chains. We have now modified this Figure to now show density corresponding to this\nregion and removed stick representations of any residues within the linker. We thank the\nreviewer for asking whether AlphaFold predicts this interaction as we had not tested this. We\nhave now added an additional Figures (Supplementary Figs. 15 and 16) and text to show\nhow AlphaFold struggles to predict the exact location of the linker, although a very rough\nregion can be identified. We also show that if the full protein and nucleosome interaction are\npredicted through AlphaFold, however, again AlphaFold cannot confidently predict where\nthe SAP and linker would bind, with perhaps one of the top 5 ranks being close to what we\nobserve experimentally. As mentioned above in response to similar comments, upon looking\nback at previous data we also observe density for the SAP domain in the H2A constructs. We\nhave now added an additional Figure (Supplementary Fig. 14) and text to explain this. We\nalso explain more clearly in the manuscript that the functional data shows it is the SAP\ndomain rather than the linker which is important for chromatin recruitment. The linker\ninteraction would be dependent on where the double-strand break would occur and is\ntherefore more likely to be unspecific binding. We have now added more text for this. With\nthis in mind to study interactions between Ku and nucleosomes would require full length\nManuscript number: NCOMMS-25-32754-T\nCryo-EM structures of NHEJ assemblies bound to nucleosomes\nprotein and nucleosome constructs. As mentioned in detail above we have tried several\nbiochemical experiments including ITC, FIDA and BLI and all have proved unsuccessful\n(Figure 1 – above). Finally, we have explored the interaction between the SAP domain and\nDNA further by expression and purification and EMSA gels and unfortunately the SAP\ndomain in isolation does not bind to DNA and therefore is difficult to study (Figure 2 –\nabove).\nMinor and editorial points:\nLine 53: Perhaps define DNA-PK as DNA-dependent protein kinase first before abbreviating\nit.\nWe thank. The reviewer and have now changed this.\nLine 161: Please indicate at which position DNA-PKcs phosphorylates H2AX.\nWe have now added this as Serine 139.\nFigure 2 A and C : These panels overlap with gel labels (“Nucleosomes”) covered by Figure\n2C.\nWe thank the reviewer as we had not noticed this and have now adjusted the figure\naccordingly.\nFigure 2 and general: Consider standardising the use of capital vs. lowercase letters.\nWe have now checked and adjusted.\nExtended Figure 2 : The local refinement depictions appear somewhat blurry (also in\nExtended Figure 5). In general, the processing scheme images are not very sharp. Perhaps\nthis is due to the downsampled review files, but if not, please consider improving their\nresolution and contrast.\nThis has now been checked and replaced.\nReferences\n1. Walker, J. R., Corpina, R. A. & Goldberg, J. Structure of the Ku heterodimer bound to\nDNA and its implications for double-strand break repair. Nature 412, 607–614 (2001).\n2. Britton, S., Coates, J. & Jackson, S. P. A new method for high-resolution imaging of Ku\nfoci to decipher mechanisms of DNA double-strand break repair. J Cell Biol 202, 579–595\n(2013).\n3. Shioi, T. et al. Cryo-EM structures of RAD51 assembled on nucleosomes containing a\nDSB site. Nature 628, 212–220 (2024).\n4. Boyer, J. A. et al. Structural basis of nucleosome-dependent cGAS inhibition. Science 370,\n450–454 (2020).\nManuscript number: NCOMMS-25-32754-T\nCryo-EM structures of NHEJ assemblies bound to nucleosomes\n5. Gerodimos, C. A., Watanabe, G. & Lieber, M. R. Nonhomologous DNA end joining of\nnucleosomal substrates in a purified system. DNA Repair (Amst) 106, 103193 (2021).\n6. Flaus, A., Luger, K., Tan, S. & Richmond, T. J. Mapping nucleosome position at single\nbase-pair resolution by using site-directed hydroxyl radicals. Proc Natl Acad Sci U S A 93,\n1370–1375 (1996)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have thoroughly addressed my initial comments/concerns, with one exception. I still believe that the cellular experiments carried out to define the functional importance of the SAP-linker in chromatin binding are underdeveloped and difficult to interpret, as was also noted by the other reviewers. Given the inability to characterize any of the SAP-linker mutants in vitro, the use of a truncated SAP-linker for the cellular experiments, and the absence of DSBs in these cellular experiments, it’s unclear how the findings connect back to the role of the SAP-linker in Ku70/Ku80 DSB recognition in chromatin that the authors thoroughly describe in their structural analysis. I do appreciate the technical difficulties the authors noted for these experiments, and so I feel it best to default to the editor to decide if this was sufficiently addressed and/or whether these experiments in their current state add any value to the manuscript. I must stress that the rest of this manuscript represents a significant advance in the DNA repair field, and I do feel the manuscript is otherwise suitable for publication in Nature Communications. I applaud the authors for taking on this challenging project.\nMinor Comments\n1. The gel in Supplementary Figure 17 appears to be mislabeled. I believe the right side of the gel should be +Ku70/80-ΔSAP.", "reviewer_2": "The revised manuscript includes changes in wording, but it lacks any new experiments necessary to assess the significance of the structural observations made with the Ku70/80 and nucleosome complexes. ED Fig. 17 does not present the binding affinities of full-length Ku70/80 and the ΔSAP variant to free DNA; instead, it demonstrates the loading of multiple Ku70/80 molecules onto a 153-bp oligo DNA. Additionally, there are no measurements of the binding affinity of Ku70/80 to nucleosomes (147 bp or 153 bp) as requested in the first round of review.\nThe following measurements are necessary for the reconsideration of the manuscript:\n(a) Kd of Ku70/80-FL and Ku70/80-ΔSAP to free DNA oligonucleotides, e.g., 20 bp.\n(b) Kd of Ku70/80-FL and Ku70/80-ΔSAP to 147-bp and 153-bp nucleosomes.\nTo avoid loading of multiple Ku70/80 molecules onto one DNA duplex, Kd can be measured with binding partners at a constant molar ratio of 1:1.", "reviewer_3": "The authors satisfactorily addressed my points. I appreciate efforts to better understand SAP domain nucleosome binding and the manuscript is revised appropriately. I am happy with the current version and can support publication."}, "author_response": "Response to Reviewers 2\nWe thank the editor and reviewers for their thorough and thoughtful evaluation of our\nmanuscript. We are encouraged by the overall positive feedback and pleased that\nthe reviewers consider our work a significant contribution to the field and suitable for\npublication. Below, we provide a detailed point-by-point response to the remaining\ncomments.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have thoroughly addressed my initial comments/concerns, with one\nexception. I still believe that the cellular experiments carried out to define the\nfunctional importance of the SAP-linker in chromatin binding are underdeveloped\nand difficult to interpret, as was also noted by the other reviewers. Given the inability\nto characterize any of the SAP-linker mutants in vitro, the use of a truncated SAP-\nlinker for the cellular experiments, and the absence of DSBs in these cellular\nexperiments, it’s unclear how the findings connect back to the role of the SAP-linker\nin Ku70/Ku80 DSB recognition in chromatin that the authors thoroughly describe in\ntheir structural analysis. I do appreciate the technical difficulties the authors noted for\nthese experiments, and so I feel it best to default to the editor to decide if this was\nsufficiently addressed and/or whether these experiments in their current state add\nany value to the manuscript. I must stress that the rest of this manuscript represents\na significant advance in the DNA repair field, and I do feel the manuscript is\notherwise suitable for publication in Nature Communications. I applaud the authors\nfor taking on this challenging project.\nWe thank the reviewer for acknowledging that the majority of the initial comments and\nconcerns have been addressed. In the revised manuscript, we have now further\ncharacterized key SAP-linker mutants in vitro and incorporated these new findings.\nUsing EMSA assays, we demonstrate that the Ku70 SAP domain binds directly to\nDNA. Notably, mutation of the conserved K596 residue results in a marked reduction\nin DNA-binding affinity, highlighting its critical role in mediating this interaction. These\nbiochemical findings support our cellular data, which also identified K596 as essential\nfor efficient Ku70/80 recruitment to chromatin.\nThese results are now presented in an updated Figure 2, along with corresponding\nadditions to the main text and Methods section. Furthermore, we provide additional\nEMSA data in Supplementary Figure 17, demonstrating the importance of T577 in\nSAP domain–DNA binding.\nMinor Comments\n1. The gel in Supplementary Figure 17 appears to be mislabeled. I believe the right\nside of the gel should be +Ku70/80-ΔSAP.\nThis has now been changed, thank you for noticing.\nReviewer #2 (Remarks to the Author):\nThe revised manuscript includes changes in wording, but it lacks any new\nexperiments necessary to assess the significance of the structural observations\nmade with the Ku70/80 and nucleosome complexes. ED Fig. 17 does not present the\nbinding affinities of full-length Ku70/80 and the ΔSAP variant to free DNA; instead, it\ndemonstrates the loading of multiple Ku70/80 molecules onto a 153-bp oligo DNA. Additionally, there are no measurements of the binding affinity of Ku70/80 to\nnucleosomes (147 bp or 153 bp) as requested in the first round of review.\nThe following measurements are necessary for the reconsideration of the\nmanuscript:\n(a) Kd of Ku70/80-FL and Ku70/80-ΔSAP to free DNA oligonucleotides, e.g., 20 bp.\n(b) Kd of Ku70/80-FL and Ku70/80-ΔSAP to 147-bp and 153-bp nucleosomes.\nTo avoid loading of multiple Ku70/80 molecules onto one DNA duplex, Kd can be\nmeasured with binding partners at a constant molar ratio of 1:1.\nWe thank the reviewer for raising the important point regarding the binding affinities of\nKu70/80 to DNA, both in the presence and absence of the SAP domain. This topic has\nrecently been addressed in the publication titled “The KU70-SAP domain has an\noverlapping function with DNA-PKcs in limiting the lateral movement of KU along\nDNA”, in which one of our co-authors, Dr. Mauro Modesti, is also a contributor. After\ndiscussion within our team, we concluded that replicating the same experiments would\nnot significantly enhance the value of our current study.\nNonetheless, to strengthen the manuscript, we have incorporated an additional\nexperiment in collaboration with Dr. Modesti. This new experiment, now included in\nFigure 2 and Supplementary Figure 17, presents an EMSA analysis showing the\nbinding of the wild-type Ku70 SAP domain to DNA. Importantly, we also demonstrate\nthe impact of two specific mutations (K596A and T577E) in the SAP domain, which\nresult in reduced DNA binding, thereby underscoring the functional relevance of these\nresidues.\nWe believe this new data provides valuable mechanistic insight into the DNA-binding\nactivity of the Ku70 SAP domain. As noted in our previous response, attempts to\ndetermine Kd values for binding to nucleosomes have proven technically challenging,\nand we were unfortunately unable to obtain reliable measurements.\nReviewer #3 (Remarks to the Author):\nThe authors satisfactorily addressed my points. I appreciate efforts to better\nunderstand SAP domain nucleosome binding and the manuscript is revised\nappropriately. I am happy with the current version and can support publication.\nMany thanks for your response."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have addressed my remaining concerns.", "reviewer_2": "The EMSA data published or presented in this manuscript support the conclusion that the SAP domain binds to DNA; however, it does not provide evidence for binding to histones. Furthermore, the mutant analysis reinforces the importance of DNA binding by the SAP domain, but it does not address whether SAP binds to chromatin proteins, contrary to the implications made by the authors.\nThe manuscript lacks data to substantiate the claim that Ku70/80 binds to nucleosomes beyond its interaction with DNA."}, "author_response": "Response to Reviewers 3\nWe thank the editor and reviewers for their continued thorough and thoughtful\nevaluation of our manuscript. We have added our comments below and now hope\nthe manuscript is ready for publication.\nReviewer #1 (Remarks to the Author):\nThe authors have addressed my remaining concerns.\nMany thanks.\nReviewer #2 (Remarks to the Author):\nThe EMSA data published or presented in this manuscript support the conclusion\nthat the SAP domain binds to DNA; however, it does not provide evidence for binding\nto histones. Furthermore, the mutant analysis reinforces the importance of DNA\nbinding by the SAP domain, but it does not address whether SAP binds to chromatin\nproteins, contrary to the implications made by the authors.\nThe manuscript lacks data to substantiate the claim that Ku70/80 binds to\nnucleosomes beyond its interaction with DNA.\nThe reviewer is correct; the SAP domain binds DNA not histones – we have added\nan additional panel to Fig 2F to show it can also bind nucleosome bound DNA and\nfree DNA. We have also modified the text throughout to make this clear.\nThe revised manuscript includes changes in wording, but it lacks any new experiments\nnecessary to assess the significance of the structural observations made with the\nKu70/80 and nucleosome complexes. ED Fig. 17 does not present the binding affinities\nof full-length Ku70/80 and the ΔSAP variant to free DNA; instead, it demonstrates the\nloading of multiple Ku70/80 molecules onto a 153-bp oligo DNA. Additionally, there are\nno measurements of the binding affinity of Ku70/80 to nucleosomes (147 bp or 153 bp)\nas requested in the first round of review.\nThe following measurements are necessary for the reconsideration of the manuscript:\n(a) Kd of Ku70/80-FL and Ku70/80-ΔSAP to free DNA oligonucleotides, e.g., 20 bp.\n(b) Kd of Ku70/80-FL and Ku70/80-ΔSAP to 147-bp and 153-bp nucleosomes.\nTo avoid loading of multiple Ku70/80 molecules onto one DNA duplex, Kd can be\nmeasured with binding partners at a constant molar ratio of 1:1.\nThe EMSA data published or presented in this manuscript support the conclusion\nthat the SAP domain binds to DNA; however, it does not provide evidence for binding\nto histones. Furthermore, the mutant analysis reinforces the importance of DNA\nbinding by the SAP domain, but it does not address whether SAP binds to chromatin\nproteins, contrary to the implications made by the authors.\nThe manuscript lacks data to substantiate the claim that Ku70/80 binds to\nnucleosomes beyond its interaction with DNA."}]

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