DNA end configurations dictate synaptic complex formation during NHEJ-mediated end bridging

Domain: chemistry
Published: 2026-01-16
Editorial outcome: major_revision

Abstract
Synapsis, the physical bridging of two broken DNA ends, is a critical step in non-homologous DNA end joining (NHEJ), the primary pathway for repairing DNA double-strand breaks (DSBs) in vertebrates. While NHEJ synapsis has been well characterized using blunt-ended DNA, how diverse DNA end configurations influence synaptic complex formation remains much less clear. Here, using single-molecule FRET, we show that end configurations play a decisive role in synapsis pathway choice, with end pairing compensating for XLF in facilitating synaptic complex transitions. Notably, 3-nucleotide (nt) microhomology at DNA ends significantly enhances close synaptic (CS) complex formation mediated by Ku70-Ku80 (Ku) and XRCC4-Ligase IV (X4L4), even in the absence of XLF. Although XLF is nonessential for synapsis with 3-nt microhomology, it further promotes CS formation when present. The FRET analysis reveals that the single-stranded and double-stranded junctions of the DNA ends are in close contact within the primary CS complex, while the overhangs flip out of the duplex, potentially stabilizing the complex. These findings underscore the pivotal role of DNA end configurations in regulating synapsis and their broader implications for NHEJ repair efficiency and fidelity.

Introduction
DNA double-strand break (DSB) is the most severe DNA damage in cells. DSB can be caused by both endogenous factors, such as active stresses from common metabolic processes, and exogenous reasons, such as genotoxic reagents1. If not repaired promptly, DSBs can result in cell cycle arrest, apoptosis, or senescence. Two major repair pathways—homologous recombination (HR) and non-homologous end joining (NHEJ)—are applied in cells to cope with DSBs2–4. HR requires homologous sequences as the template for repair; therefore, it mainly has roles in the S and G2 phases of the cell cycle. NHEJ can directly rejoin the broken DNA ends with no to little homology and can function throughout the entire cell cycle5,6. Because HR copies sequences from homologous templates, it can largely restore the original DNA sequences4. NHEJ, however, is more error-prone than HR, and often leaves an ‘information scar’ after repair2,3. However, many mammalian somatic cells exist in G0 for a large fraction of the time, and NHEJ, therefore, is the predominant pathway for repairing DSBs within cells7.

One of the most intriguing and important stages in this major DNA repair pathway is bringing the two DNA ends at a DSB together into physical proximity—a process called synapsis3,8. The essential components required for NHEJ synapsis have been identified by varied single-molecule techniques9–15 using blunt-ended DNA substrates. A two-structure synaptic model has been proposed for NHEJ synapsis using both purified NHEJ proteins9 and Xenopus crude extracts11. The studies using purified NHEJ proteins show that Ku70-Ku80 (Ku) and XRCC4-DNA ligase IV (X4L4) are sufficient to mediate NHEJ synapsis of blunt ends to form a flexible synaptic (FS) complex, in which the DNA molecules are aligned laterally near the end, and the two DNA ends can loosely search for base pairing within one another; XRCC4-like factor (XLF) and/or Paralog of XRCC4 and XLF (PAXX) facilitates the transition from FS to a close synaptic (CS) state, in which the DNA ends are positioned end-to-end, allowing for ready ligation9. Importantly, Ku, X4L4, and XLF/PAXX can mediate CS formation in either a two-step or a one-step manner9. Other studies using Xenopus crude extracts suggest a two-stage synapsis mechanism, in which Ku and DNA-PKcs first form a long-range synaptic complex; XLF, non-catalytic X4L4 activity, and DNA-PKcs kinase activity are then required to mediate the transition from long-range synapsis to short-range synapsis. The two DSB ends in the long-range synaptic complex are 115 Å apart, while the ends are in close contact in the short-range synaptic complex and ready for ligation11. Although discrepancies exist for these different studies using different systems9,11, the core NHEJ proteins—Ku, X4L4, and XLF—are required for NHEJ synapsis and transitions of synaptic complexes, and these proteins mainly function close to the ends of the broken DNAs8,9,11.

To date, almost all the studies investigating NHEJ synapsis primarily used blunt-ended DNA substrates9–11, which could allow identification of the essential NHEJ components contributing to synapsis. However, blunt ends are a small portion of the spectrum of DNA end configurations at the DSBs generated endogenously and exogenously3,16–18. Most DSB ends have diverse configurations and structures, and some even have damaged bases or nucleotides19,20. Previous studies unveil that the end configurations determine which NHEJ proteins are required for the timely repair of DSBs and the consequences of the repair products21. Moreover, damaged bases and mispaired ends were reported to increase the mobility of DNA ends within the synaptic complexes mediated by Ku, X4L4, and XLF proteins, highlighting the importance of end mobility in NHEJ repair19. While DNA end configurations play important roles in NHEJ repair and may also in the synapsis step, it is unclear if DNA end configurations affect the two structurally different synaptic complexes observed with blunt-ended DNA substrates9, and also not clear if the end configurations could determine the choice of NHEJ proteins for DNA synapsis and the structures of formed synaptic complexes, namely the alignments of the two DNA molecules within the synaptic complex. Moreover, it is unclear if different end configurations influence FS which first brings two distant DSBs together, and CS which mainly adjusts the local realignments of two dsDNA at the ends.

Here, using single-molecule FRET (smFRET), we directly evaluate the effects of DNA end configurations on NHEJ synapsis by core NHEJ proteins – Ku, X4L4, and XLF, and investigate the structures of synaptic complexes and the dynamics of synapsis determined by the end configurations. We find that DNA end structures critically influence both the efficiency of NHEJ synapsis and the architecture of the synaptic complexes formed. The presence of 3-nucleotide (nt) microhomology at DNA termini promotes efficient CS complex formation by Ku and X4L4 even in the absence of XLF. While XLF is not essential for close synapsis of ends with 3-nt microhomology, it enhances the efficiency of CS complex. FRET analyses of the synaptic complexes reveal that the single-stranded (ss) and double-stranded (ds) junctions of the overhangs are in close contact in the synaptic complex, while the overhangs themselves form flaps at the DNA ends. These flapped overhangs may facilitate base pairing, thus stabilizing the CS complex mediated by Ku and X4L4, and may also facilitate end processing by NHEJ nucleases and polymerases. Our findings underscore the critical role of DNA end configurations in determining NHEJ synapsis, highlighting their importance across all stages of NHEJ repair.

Results
DNA end configurations influence NHEJ synapsis
Using blunt-ended DNA, we found that Ku70-Ku80 (Ku) together with XRCC4-DNA ligase IV (X4L4) efficiently mediate NHEJ synapsis, forming a flexible synaptic (FS) complex, which is characterized as having a FRET peak around 0.1; and some of the formed synaptic complexes exhibit FRET fluctuations, indicating the mobility of the two dsDNA molecules within the FS complex (Supplementary Fig. 1a–e). XLF promotes the formation of a close synaptic (CS) complex, which is characterized as exhibiting a FRET peak around 0.8–similar to that of the pre-ligated substrates–indicating end-to-end contact of the DNA molecules (Supplementary Fig. 1c, f, g). Dwell-time analysis of the formed synaptic complexes showed lifetimes of 9.9–16.5 s for the FS complex and 29.9 s for the CS complex formed in the presence of XLF (Supplementary Fig. 1h–j). These results are consistent with our previous study9, indicating the accuracy of the smFRET system and assay developed here for probing NHEJ synapsis.

The structural features of FS and CS complexes observed for blunt ends suggest that the two ends can be covalently ligated only when a cofactor of X4L4, e.g., XLF, is present in the reaction, which is confirmed by the ensemble ligation assay (Fig. 1a, lane 3). In contrast, compatible overhangs can be directly ligated by Ku plus X4L4 without XLF, with efficiency comparable to that with XLF present (57.5% vs 59.7%) (Fig. 1a, lanes 5 and 6). This indicates that the overhang complementarity can substitute for XLF in producing a ligation-competent CS complex. Moreover, X4L4 alone can ligate compatible overhangs (Supplementary Fig. 1k), further supporting a role for overhang pairing in CS formation.

To directly test the effect of overhangs on NHEJ synapsis, we prepared duplexes with 3’ four-nucleotide complementary overhangs (GGGG/CCCC) for smFRET analysis (Fig. 1b). Both substrates have 5’ OH groups at the ends; therefore, we can focus only on synapsis but not ligation. Ku plus X4L4 can mediate the synapsis of the 3’ GGGG/CCCC overhangs (Fig. 1c and Supplementary Fig. 1l). The lifetimes of synaptic complexes formed by Ku plus X4L4 with these overhangs were similar to those for blunt ends (13.2 vs 16.0 s) (Supplementary Fig. 1h, m), but their synapsis efficiency was ~2.5-fold higher (Fig. 1d). In contrast to blunt ends, where XLF markedly increases synapsis efficiency9, XLF had little effect on the synapsis of 3’ GGGG/CCCC overhangs (Supplementary Fig. 1n). Thus, compatible overhangs can compensate for XLF’s role in promoting synapsis, underscoring the influence of DNA end configuration on NHEJ.

Compatible overhangs promote XLF-independent CS formation by Ku plus X4L4
We next examined the structures of synaptic complexes formed by Ku plus X4L4 with 3’ GGGG/CCCC overhangs, focusing on the spatial arrangement of the overhangs within synaptic complexes. FRET trajectories revealed four distinct states with approximate levels of 0.75, 0.55, 0.4, and <0.2 (Fig. 2a and Supplementary Fig. 2a). Corresponding FRET distributions exhibited four obvious peaks, which have the Gaussian-fitted peaks at 0.76, 0.58, 0.39, and 0.13, respectively (Fig. 2b). The highest and lowest peaks match those observed for FS and CS complexes formed with blunt ends in the presence of Ku, X4L4 and XLF (Supplementary Fig. 1f). This indicates that the duplexes within these two types of synaptic complexes formed with 3’ GGGG/CCCC overhangs and with Ku plus X4L4 are organized in a similar way to that in the corresponding complexes with blunt ends and with Ku plus X4L4 plus XLF, namely Ku plus X4L4, without XLF, can promote efficient FS and CS formation when DNA ends have compatible overhangs. These findings suggest that compatible overhangs not only promote CS formation but also facilitate the FS-to-CS transition mediated by Ku and X4L4. These findings suggest that compatible overhangs not only promote CS formation but also facilitate the FS-to-CS transition mediated by Ku and X4L4.

Similar results were obtained for ACTG/CAGT overhangs (Supplementary Fig. 2b–e), including the FRET states of the trajectories (Supplementary Fig. 2d), the FRET distributions of formed synaptic complexes (Supplementary Fig. 2e), and minimal effect of XLF on synapsis efficiency (Supplementary Fig. 2c). These findings indicate that the ability of Ku plus X4L4 to promote CS formation is governed primarily by overhang compatibility rather than by base composition. The A/T-rich TTTT/AAAA overhangs likewise produced similar FRET distributions (Supplementary Fig. 2f), demonstrating that Ku plus X4L4 can support both FS and CS formation even for A/T-rich ends. However, the highest-FRET peak was less frequent than in G/C-rich or ACTG/CAGT overhangs, and in contrast to the G/C-rich ends, XLF enhanced synapsis efficiency for A/T-rich overhangs (Supplementary Fig. 2g), likely reflecting the lower pairing stability of A/T-rich sequences. These observations suggest that base composition modulates synapsis efficiency. Nevertheless, complementarity—rather than base composition—remains the primary determinant of synapsis, with base composition acting only as a secondary modulator.

Two structurally different CS complexes formed for DNA with compatible overhangs
Next, we examined how compatible overhangs are aligned within synaptic complexes to facilitate the formation of, or transition to, the CS complex and to stabilize it. To establish reference FRET values, we prepared several duplex DNA substrates labeled with Cy3 and Cy5 dyes at different distances (Fig. 2c and Supplementary Fig. 2h). These duplexes were generated by pre-ligating either 3’ overhang substrates with different lengths of G/C pairs or blunt-end substrates. Because the Cy3 and Cy5 dyes are positioned 4 bp from the ssDNA–dsDNA junctions on all 3’ overhang substrates, a direct in-line junction–junction contact between the 3’ GGGG/CCCC overhangs results in an 8-bp Cy3–Cy5 separation (Fig. 2d, E4). Full pairing of the GGGG and CCCC overhangs produces a 12-bp separation with two nicks at the junctions (Fig. 2d, E3), whereas partial in-line pairing generates a 13–15-bp separation with two gaps between the overhangs (Fig. 2d, E3). For pre-ligated blunt ends, the Cy3-Cy5 dyes are separated by 8 bp, producing a FRET at 0.76. Therefore, this configuration can serve as a reference for a junction-contacting synaptic complex in which the two duplexes are aligned in-line, and the dsDNA junctions are in direct contact (Fig. 2d, E4). For overhang substrates to adopt a junction-contacting configuration, the ssDNA overhangs must project outward from the duplex axes to allow dsDNA junctions to touch (Fig. 2d). Pre-ligated compatible GGGG/CCCC and GGGGGG/CCCCCC overhangs have dye separations of 12bp and 14bp, respectively. These mimic synaptic complexes in an in-line alignment where the overhangs are base-paired, leaving either two nicks (nick-containing configuration) or two gaps (gap-containing configuration) between them (Fig. 2d, E3). Because the ends consist of repeated G/C bases, the overhangs can slide relative to each other, allowing partial pairing and transitions between nick-containing and gap-containing states. The corresponding FRET peaks (~0.57 and ~0.52) are close enough to be indistinguishable in our system (Fig. 2c); therefore, we collectively refer to them as the end-pairing configuration.

When 3’ GGGG/CCCC overhangs form synaptic complexes, the most abundant species is the E4 peak (~0.76; Fig. 2b), matching the pre-ligated blunt-end reference (Fig. 2c). This indicates a junction-contacting configuration in which the ssDNA overhangs flip outward (Fig. 2d, E4). The next FRET peak, E3 (~0.58), resembles the pre-ligated GGGG/CCCC and GGGGGG/CCCCCC references, suggesting an end-pairing configuration (including both nick-containing and gap-containing states). Lower FRET peaks, E2 (~0.39) and E1 (~0.13), are smaller than the referenced duplex with a 14bp Cy3-Cy5 separation (Fig. 2c), suggesting that the overhangs may be in a gap-containing state with only distal G/C base pairing. However, pairing of only a single G/C base is insufficient for Ku and X4L4 to stabilize an end-pairing CS complex (see next section). Thus, the E2 and E1 states represent a looser alignment resembling the FS complex formed by blunt ends (Fig. 2d and Supplementary Fig. 1c).

Since the CS complex formed by blunt ends and by Ku, X4L4, and XLF has close-contact ends, and synaptic complexes of the overhang substrates corresponding to both E3 and E4 states involve end-to-end contact, either at the ssDNA overhangs (E3) or at the ssDNA–dsDNA junctions (E4), we classify the E4 and E3 complexes also as CS complexes. The end-pairing CS complex (E3) can form de novo (Fig. 2a, top panel), or via transition from either the FS complex (Fig. 2a, bottom panel; Supplementary Fig. 2a, left and middle panels) or the junction-contacting CS complex (E4) (Supplementary Fig. 2a, left and middle panels). In contrast, the junction-contacting CS complex typically arises from the end-pairing CS complex rather than directly from the FS complex (Fig. 2a and Supplementary Fig. 2a). These results highlight the central role of end-pairing in CS formation mediated by Ku and X4L4.

Compatible ACTG/CAGT overhangs also yield both junction-contacting (E4) and end-pairing (E3) CS complexes (Supplementary Fig. 2d, e, i), which excludes the effects of repetitive bases on the formation of CS. Therefore, the results above indicate that at least two structurally distinct CS complexes (junction-contacting and end-pairing) can form for DNA substrates with compatible overhangs. The pairing of the overhangs can compensate for XLF’s role in promoting CS complex formation as required in blunt-end synapsis. The (potential) pairing of the end overhangs either as in the junction-contacting CS complex or the end-pairing (nick-containing and gap-containing) CS complex can stabilize the CS complex formed by Ku plus X4L4.

We further tested whether the common oxidative lesion 8-oxoguanine affects synapsis mediated by Ku plus X4L4. 3’ GGGG/CCCC substrates containing one or two 8-oxoguanines at defined positions were designed (Fig. 2e). A single lesion did not impair synapsis efficiency, whereas two lesions caused only a modest reduction (Fig. 2f and Supplementary Fig. 3). Neither one nor two lesions disrupted CS formation or stability (Fig. 2g, i). Thus, 8-oxoguanine has minimal impact on synapsis—including synapsis efficiency and CS formation—mediated by Ku plus X4L4, consistent with previous findings that NHEJ can tolerate 8-oxoguanine within the overhangs at DNA ends19,22.

A minimum of three complementary base pairs are required for efficient CS formation by Ku and X4L4
We next asked how many complementary base pairs are needed to promote CS by Ku and X4L4. To address this, we tested DNA substrates with compatible ends ranging from one (G/C) to three (GGG/CCC) base pairs (Fig. 3a). Single-molecule images and trajectories confirmed that Ku and X4L4 can mediate synapsis of all these substrates (Fig. 3b–e). However, synapsis of ends with one (G/C) or two (GG/CC) base pairs yielded primarily low FRET signals (<0.5) (Fig. 3c, d), similar to blunt ends (Supplementary Fig. 1e). Corresponding FRET distributions of all the synaptic complexes for G/C as well as GG/CC overhangs are also similar to that of blunt ends. The main peaks of FRET distributions are around 0.11 and 0.09, respectively (Fig. 3f, g), which are substantially lower than those of pre-ligated overhangs (Supplementary Fig. 4a). Thus, Ku and X4L4 are not able to proceed beyond FS complexes for substrates with ≤2 base pairs, suggesting that two base pairs at ends do not provide sufficient energy for the FS-to-CS transition.

By contrast, overhangs with three base pairs (GGG/CCC) produced high FRET traces (Fig. 3e), with FRET distributions similar to those of GGGG/CCCC substrates (Figs. 2b, 3h). These results indicate that Ku and X4L4 can promote efficient CS complexes formation, including junction-contacting complex (E4) and end-pairing complex (E3), for GGG/CCC overhangs (Supplementary Fig. 4b), thus at least three base pairs between overhangs are needed to provide the energy for efficient FS-to-CS transition and to stabilize the formed CS. The quantification of FS and CS also showed that CS occurs only for overhangs with ≥3 base pairs (Fig. 3i), thus indicating a minimum of three base pairs between ends is required for efficient CS formation by Ku and X4L4. Although synapsis efficiency did not change significantly between one- and two-base pair overhangs, efficiency increased markedly when complementarity reached four base pairs (Fig. 3j). This indicates that extending microhomology not only promotes synapsis efficiency but also favors CS formation, likely through overhang pairing that stabilizes the transition from FS to CS. Nevertheless, the stability of the CS complex as reflected by the duration exhibited insignificant differences between GGG/CCC and GGGG/CCCC substrates (Supplementary Fig. 4c), suggesting that three base pairs are sufficient to form a stable CS.

To further assess the contribution of paired overhangs, we varied the length of the overhangs on immobilized substrates (0–3 Gs) while maintaining four complementary Cs on the solution substrate (Supplementary Fig. 4d). The results showed that an obvious CS complex corresponding to the FRET peak of E4 were observed only when three complementary Gs were present on the immobilized end (Supplementary Fig. 4e–i), and the dwell times of CS complexes were comparable between GGG/CCC and GGGG/CCCC overhangs (Supplementary Fig. 4c, j). We also examined whether Ku and X4L4 can promote CS formation when the three-base-pair microhomology is embedded within longer, partially incompatible overhangs (GGGTT/CCCTTT; Fig. 3k–n and Supplementary Fig. 5). This scenario is relevant for understanding synapsis prior to end processing, such as Artemis-mediated trimming. Ku and X4L4 did promote CS formation on GGGTT/CCCTTT overhangs (Fig. 3l, m); however, both CS stability and formation efficiency were reduced relative to GGG/CCC (dwell: 4.2 s vs. 10.3 s; CS fraction: 19.6% vs. 34.8%; Fig. 3i, m, n and Supplementary Fig. 4c). These results show that three embedded complementary base pairs are sufficient for CS formation, but incompatible flanking sequences diminish the efficiency and stability of the complex.

These findings demonstrate that a minimum of three complementary base pairs is required for Ku and X4L4 to promote efficient and stable CS formation.

XLF promotes CS formation from overhangs with limited complementarity
XLF, an accessory factor of X4L423, promotes ligation by facilitating the FS-to-CS transition9. To test its role in overhang synapsis, we applied the same DNA substrates as in Fig. 3 (Fig. 4a). The results showed that increasing the number of complementary base pairs enhanced synapsis in the presence of XLF (Supplementary Fig. 6a, b), indicating that base pairing contributes to synapsis even when XLF is present. For single-base overhangs (G/C), XLF enabled formation of high FRET complexes with a peak at 0.68 (Fig. 4b), comparable to pre-ligated G/C overhangs (Supplementary Fig. 4a) and within the range of blunt-end CS (Fig. 2c). Similarly, GG/CC overhangs displayed high FRET peaks in the presence of XLF (Fig. 4c). Notably, the highest FRET peak for GG/CC (0.68) was larger than that of pre-ligated GG/CC (0.64) (Fig. 4c and Supplementary Fig. 4a), consistent with formation of junction-contacting CS complexes. These results demonstrate that XLF is required for CS formation when microhomology is limited to fewer than three base pairs. For longer overhangs (GGG/CCC and GGGG/CCCC), the FRET distributions exhibited predominant high FRET peaks of 0.72–0.73 (Fig. 4d, e), which are close to that of the blunt-end CS complex (Fig. 2c). This suggests that XLF further promotes junction-contacting CS complexes, likely through interactions with Ku and X4L4 as previously reported9. Moreover, across all substrates, XLF increased the proportion of CS complexes relative to reactions without XLF (Fig. 4b–f vs Fig. 3f–i), with CS abundance correlating with overhang length (Fig. 4f). The results above demonstrate that base pairing between the overhangs and XLF contributes to CS complex formation.

Dwell-time analysis revealed no significant difference between CS complexes formed with or without XLF, or among different overhang lengths (Fig. 4g). The results suggest that base pairing between overhangs can supplement the bridging role of XLF in promoting CS formation, and both aspects contribute to CS complex formation, thus leading to a higher amount of CS for overhangs with longer microhomologies (Fig. 4f). In addition, XLF substantially enhanced both the formation and stabilization of CS complexes for GGGTT/CCCTT overhangs (Supplementary Fig. 5b and Fig. 4h–j), indicating that XLF also plays a key role in synapsis of partially compatible overhangs.

Together, these results above demonstrate that XLF can promote CS complex formation across substrates, especially for overhangs with limited microhomology.

DNA end chemistry influences CS formation
It has been reported that synapsis can also be modulated by end chemistry, particularly the presence of 5’ phosphate groups (5’ P), which facilitates synapsis and stabilize complexes formed by Ku, X4L4, and XLF24. To examine this in the context of overhangs by Ku and X4L4, we prepared DNA substrates with 5’ P at both ends (Fig. 5a). The results showed that synapsis efficiency increased significantly for overhangs longer than one base pair when 5’ P was present (Fig. 3j and Supplementary Fig. 7a, b).

With 5’ phosphorylated G/C overhangs, FRET distributions showed only low peaks (E1 = 0.11 and E2 = 0.3), suggesting the formation of FS complexes (Fig. 5b). In contrast, 5’ phosphorylated GG/CC overhangs yielded three dominant peaks (E2 = 0.16, E3 = 0.51, and E4 = 0.72) (Fig. 5c), suggesting the formation of FS (E2), nicking-containing CS (E3), and junction-contacting CS (E4) complexes. A single 5’ phosphate at one end also promoted CS formation to a similar extent as two phosphorylated ends (Supplementary Fig. 7c–h), although dual phosphorylation provided a modest additional enhancement for GG/CC overhangs (Fig. 5e and Supplementary Fig. 7h). The formation of CS complexes for 5’ phosphorylated GG/CC overhangs indicates that both base pairing (two nucleotides) and 5’ P act cooperatively to promote CS formation. Because GG/CC overhangs without 5’ P primarily formed FS (Fig. 3g) and G/C overhangs even with 5’ P failed to form CS (Fig. 5b). The interactions between 5’ P and adenylated Ligase IV25,26 may help stabilize and transition intermediates toward CS. 5’ P also promoted CS formation with GGG/CCC substrates (Figs. 3h–i, 5d, e). Moreover, duplexes synapsed within CS complexes from GG/CC and GGG/CCC overhangs were resistant to 2 M NaCl (Fig. 5f), indicating covalent ligation of the duplexes by X4L4. This was also confirmed by ensemble ligation assays, which showed successful ligation of these overhangs by Ku and X4L4 (Supplementary Fig. 8). These results reveal that CS complexes formed with overhangs by Ku and X4L4 are ligation-competent. The ligation likely occurs directly within the nick-containing CS complexes or following transitions from junction-contacting to nick-containing CS states.

Together, these findings show that DNA end chemistry, specifically 5’ phosphorylation, enhances both synapsis efficiency and the formation of ligation-competent CS complexes.

Methods
DNA substrates
The oligonucleotides used in this study were synthesized and purified by Sangon Biotech (Shanghai, China). Their sequences are listed in Supplementary Data 1. Duplexes were prepared by annealing corresponding oligonucleotides (Supplementary Table 1) in the annealing buffer of 20 mM Tris-HCl (pH 8.0) and 100 mM NaCl. The double-stranded DNA (dsDNA) substrates immobilized on the slide include 5’OH/P-BZ15/HC120, 5’OH/P-BZ15/HC124, 5’OH/P-BZ15/HC123, 5’OH/P-BZ15/HC122, 5’OH/P-BZ15/HC116, 5’OH/P-BZ15/CZ70, 5’OH/P-BZ15/BZ80, 5’OH/P-BZ15/CZ72, 5’OH/P-BZ15/CZ93, 5’OH/P-BZ15/CZ94, 5’OH/P-BZ15/CZ95, 5’OH/P-BZ15/CZ87 and 5’OH-BZ15/HC119, in which the oligonucleotides HC120, HC124, HC123, HC122, HC116, HC119, CZ70, CZ72, CZ93, CZ94, CZ95 and CZ87 contain a biotin at their 5’ end for immobilization. The duplexes used in solution include 5’OH/P-BZ35/(BZ52+BZ24), 5’OH/P-BZ35/(BZ71+BZ24), 5’OH/P-BZ35/(BZ55+BZ24), 5’OH/P-BZ35/(BZ70+BZ24), 5’OH/P-BZ35/(BZ36+BZ24), 5’OH/P-BZ35/(BZ81+BZ24), 5’OH/P-BZ35/(CZ69+BZ24), 5’OH/P-BZ35/(CZ71+BZ24), 5’OH/P-BZ35/(CZ92+BZ24) and 5’OH/P-BZ35/(CZ89+BZ24). These solution duplexes contain a loop at one end to let synapsis only happen at the free end. 5’OH/P-BZ35 and BZ24 first annealed with BZ52, BZ71, BZ55, BZ70, BZ36, CZ69, CZ71, CZ92 and CZ89, respectively, to form a loop- and nick-contained duplex. The nicks were then sealed by T4 DNA ligase (Sangon Biotech, Shanghai) at 15 °C overnight. Positive control substrates were prepared by ligating 5’P-BZ15/HC120 with 5’P-BZ35/(BZ52+BZ24), 5’P-BZ15/CZ72 with 5’P-BZ35/(CZ71+BZ24) and 5’P-BZ15/HC116 with 5’P-BZ35/(BZ36+BZ24), respectively. The ligation products were then resolved by 10% urea-denaturing PAGE gels and the target products on the gels were then sliced and further purified.

Protein expression and purification
Ku70/80, X4L4, and XLF were expressed and purified following the previously described protocols9 (Supplementary Fig. 1a). Briefly, Ku70/80 was expressed in High Five insect cells (Invitrogen, B855-02) and purified using Ni-NTA affinity beads (Union-Biotech, Shanghai), HiTrap Heparin HP column (Cytiva, Sweden AB, 17040601), HiTrap Capto Q ImpRes anion exchange column (Cytiva, Sweden AB, 17547055), and Superdex 200 Increase column (Cytiva, Sweden AB, 28990944). Ligase IV was co-expressed with XRCC4 in High Five insect cells and purified using Ni-NTA affinity beads, anion exchange column, and Superdex 200 column. XLF was expressed in 293T cells (ATCC, CRL-3216) by transfecting the cells with XLF cDNA contained pcDNA6 plasmid (pcDNA6/huXLF-Myc-His). After expression, XLF was purified via Ni-NTA affinity chromatography and anion exchange chromatography. The concentrations of purified recombinant proteins were measured by a 12% SDS-PAGE using BSA as the standard. Purified proteins were aliquoted and stored at −80 °C after flash-frozen using liquid nitrogen.

Ensemble NHEJ assay
The ensemble NHEJ reaction mixture included 40 nM each of the two dsDNA substrates, 1 mM ATP, 2 mM DTT, 50 nM Ku70/80, and 100 nM X4L4, with or without 100 nM XLF. The ligation buffer consisted of 25 mM Tris-HCl (pH 8.0), 75 mM KCl, and 10 mM MgCl2. Samples were incubated at 37 °C for 1 h, followed by phenol extraction and ethanol precipitation. Products were separated by 12% native PAGE and visualized using a ChemiDoc MP Imaging System with parameters for Cy5 excitation and imaging.

Slide and coverslip modification
Slides and coverslips were modified with biotin-PEG (Laysan Bio, biotin-PEG-SVA-5000) and mPEG (Laysan Bio, mPEG-SVA-5000). They were first soaked in 1 M KOH for 20 min, rinsed with purified water, and treated with a 75% H2SO4/25% H2O2 solution for 20 min. After rinsing with purified water and washing with methanol, slides and coverslips were silylated with a methanol solution containing 2% (v/v) APTES (Millipore Sigma, 281778) and 5% (v/v) acetic acid. After rinsing and drying, they were PEGylated overnight at room temperature with mPEG or a mixture of mPEG and biotin-PEG. Finally, they were washed, dried, and stored at −20 °C in a sealed container until use.

Single-molecule setup
The single-molecule FRET (smFRET) experiments were carried out on the Olympus IX83P2ZF inverted microscope equipped with the 532 nm (COHERENT, OBIS 532 nm LS 50 mW) laser. The output laser was collimated and reflected by a mirror (Chroma ZT405/488/532/640rpc-XT) into an oil immersion objective (Olympus, 100×, NA = 1.5) to achieve the TIRF illumination. The laser power was set at 1.5 mW before the objective lens. A trigger box, which is based on the UNO board from Arduino and is provided by FluoCa Scientific (Shanghai, China), was applied to synchronize the laser excitation and the camera exposure during imaging. The Cy3 and Cy5 emission wavelengths passed through a band pass filter (Chroma ZET405/488/532/642m) mounted in the microscope, and were further separated by a dichroic beam splitter (Semrock FF640-FDi01-25×36) mounted in the OptoSplit II (CAIRN RESEARCH). The Cy3 and Cy5 emissions were finally imaged on the two halves of a back-illuminated sCMOS camera (Teledyne Photometrics, Prime 95B) after passing the band pass filters of ET600/50m (Chroma) and ET700/75m (Chroma), respectively. The smFRET system was controlled, and the images were captured both with the Micro-Manger 2.0 software34,35.

Single-molecule imaging
The smFRET strategy used for the NHEJ synapsis investigation is similar as previously described9,32. Briefly, two duplexes (74–85 bp) with varied end configurations were prepared with Cy3 or Cy5-labeled at a distance of 4 bp from the double-stranded (ds) terminus and close to the single-stranded (ss) overhang. The Cy5-labeled dsDNA is immobilized on the slide, the Cy3-labeled dsDNA is free in solution and has a loop at the opposite side to the Cy3 dye. The simultaneous appearance of Cy3 and Cy5 signals indicate the occurrence of synapsis, and the changes in FRET (EFRET) signals reflect the dynamic interactions between two dsDNA ends.

The 5’OH/P-dsDNA was immobilized on the surface of the coverslips by a biotin-streptavidin-biotin strategy at a concentration of 300 pM. 10 nM loop-containing and Cy3-labeled 5’OH/P-dsDNA was used in the solution. Unless otherwise specified, the reaction buffer in this study contained 20 mM Tris-acetate pH 7.5, 75 mM KCl, 10 mM MgCl2, 3% glycerol, 1 mg/ml BSA, 1 mM DTT, 0.5 mM ATP, 0.25% Tween 20, 2 mM Trolox (Millipore Sigma, 238813) and an oxygen-scavenging system, which contains 0.8% (w/v) D-glucose, 1 mg/ml glucose oxidase (Millipore Sigma, G2133-50KU), and 0.04 μg/ml catalase (Millipore Sigma, C3556). The final concentrations of the proteins were 50 nM Ku, 100 nM X4L4, and 100 nM XLF. Proteins and looped dsDNA were added to the reaction buffer, mixed, and immediately injected into the imaging chamber. Images were captured immediately after sample injection. All reactions were carried out at room temperature (22 °C). The exposure time of the CCD camera was set to 100 ms and each movie lasted approximately 2 min, with at least three movies taken for each sample. Each sample was repeated at least three times in this study. In all, 532 nm laser was used constantly to excite FRET pairs during each ROI imaging session. In all, 532 nm laser cannot directly excite the Cy5 dye, the Cy5 dye only brightens when it is exposed to energy transferred from the excited Cy3 dye. The appearance of both Cy3 and Cy5 signals or the FRET signal indicates the occurrence of synapsis. The emitted fluorescence signals of Cy3 and Cy5 were split into two channels using a splitter (FF640-FDi01-25×36, Semrock). The Cy3 and Cy5 imaging channels were registered using the BZ45/Zhao BZ49 dsDNA probe, which contains Cy3 and distance-rational Cy5 dyes.

smFRET data analysis
FRET pair extraction and further data analysis were similar to our previous study9. Briefly, FRET pairs were searched and filtered by scanning every 20 frames of the movie using the iSMS v2.01 software built-in Matlab36,37. Since the slide surface was not completely blocked, Cy3-dsDNA in solution may adsorb non-specifically to the slide surface, resulting in some spots of Cy3 signals on the images (called Cy3-only spots). Only molecules with paired Cy3 and Cy5 signals were selected, and synapsis events were considered as molecules with paired Cy3 and Cy5 signals. All identified trajectories were then exported, and the data were further analyzed using programs written in MATLAB, as described previously9. The synapsis efficiency was measured from the number of events obtained from the reaction of 50 nM Ku and 100 nM X4L4 or the reaction of 50 nM Ku, 100 nM X4L4 and 100 nM XLF as specified in the figures. Synapsis efficiency was normalized by the number of synaptic molecules from the synapsis of blunt ends under the same condition. All molecules of each sample were combined and used to plot the EFRET histograms, and all EFRET histograms were fitted with the Gaussian distribution functions.

FS and CS complexes were distinguished based on FRET levels observed in synapsis events. Classification was guided by the full width at half maximum (FWHM) of FRET distributions obtained from pre-ligated DNA substrates. Specifically, the pre-ligated blunt-end substrate—with an 8-nt Cy3–Cy5 separation—was used as a reference to identify junction-contacting CS complexes. Because 2-nt microhomology is insufficient for stable CS formation by Ku and X4L4, the highest FRET cutoff (0.66) derived from pre-ligated 3’ GGG/CCC overhangs (peak FRET=0.60; FWHM=0.11; Supplementary Fig. 4a) was also adopted as a threshold for defining junction-contacting CS. Thus, synapsis events with FRET≥0.66 were classified as junction-contacting CS complexes. This cutoff is consistent with the lowest cutoff (0.69) observed for pre-ligated blunt ends (Supplementary Fig. 1g or Fig. 2c), which also defines junction-contacting CS complexes. The gap-containing CS complex formed by 3’ GGGG/CCCC overhangs was expected to yield the lowest CS FRET due to the 13-nt Cy3–Cy5 separation. This configuration was modeled using pre-ligated 3’ GGGGG/CCCCC overhangs (Supplementary Fig. 2h), which gave a cutoff of 0.48 at FWHM. Accordingly, synapsis events with FRET>0.48 were categorized as CS (including gap-containing, nick-containing, and junction-contacting CS), whereas events with FRET≤0.48 were classified as FS. This 0.48 threshold is further supported by FRET histograms of 3’ G/C and 3’ GG/CC overhangs (Fig. 3f, g), which form only FS complexes with Ku and X4L4 and predominantly exhibit FRET below 0.48. Following classification, the formation efficiency and dwell times of FS and CS complexes were calculated for each DNA substrate set.

Synapsis time analysis
The dwell-time analysis was based on the time window from the appearance to the disappearance of each FRET pair of molecules. All the dwell-time histograms and the corresponding exponential fits corresponded only to synapsis events that begin and end within the detection time window. Distributions were fitted with single-exponential decay functions.

Quantification and statistical methods
The Gaussian fitting of the EFRET distribution and the exponential fitting of the dwell time distribution were performed using OriginLab Origin 2024b. The statistical analysis was performed using the unpaired two-tailed t test built-in Excel and statistical significance was considered when the P value was less than 0.05. Statistical analysis was performed with at least three independent experiments.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is a review of NCOMMS-25-67092. In this study, the authors use a previously described biophysics approach for\nmeasuring single molecule DNA synapsis by core factors in the non-homologous end joining pathway. The experiments are\nstraightforward with appropriate controls. The main finding is that adding compatible DNA overhangs enhances synapsis via\nKU/XRCC4/LIG4 and reduces the requirement for XLF. My concern is that the relative requirements for NHEJ factors for\ndistinct end configurations have been relatively well established, and as such these findings are not very surprising. One\nexample is this paper from 2016 (PMID: 27703001). Much of the research on end synapsis, including from this group,\nfocuses on blunt DNA ends merely because that end configuration needs to be stabilized without annealing. Other studies\nhave also addressed the impact of non-homologous tails too. The authors mention that other end configurations are likely\nfound in cells, such as damaged/oxidized bases, but such end configurations were not tested here, but rather\nfocus on microhomology substrates. Perhaps some experiments with other such substrates would improve the scope of the study.", "reviewer_2": "In this manuscript, Zhang et al. present an elegant and well-executed study investigating the role of DNA end configurations\nin NHEJ-mediated synapsis. Using a combination of biochemical and single-molecule FRET analyses, the authors\nconvincingly demonstrate that DNA end configurations—including terminal sequences and 5′ phosphate groups—play\ncritical roles in determining the sub-pathway choice of NHEJ synapsis. Given that broken DNA ends generated during\nchemo-/radiotherapy, CRISPR-Cas9-mediated gene editing, V(D)J recombination, and class-switch recombination exhibit\ndiverse end structures, understanding how these configurations influence NHEJ synapsis has significant implications for\ngenome stability, gene editing, and antibody maturation. This makes the work highly relevant to the readership of Nature\nCommunications. I am generally supportive of this study, but several concerns should be addressed before acceptance.\nMajor concerns:\n1. The authors should provide gel images or Western blots to demonstrate the quality of the recombinant Ku70–Ku80,\nXRCC4–LIG4, and XLF proteins. Additionally, activity assays of the purified proteins should be included, as the purity and\nfunctional integrity of these proteins are crucial for supporting the conclusions drawn in this study.\n2. In Figure 5, the authors examine the effects of 5′ P on NHEJ synapsis using duplexes with 5′ phosphorylated ends on both\nstrands. The authors should also test the effect of a single 5′ P group on synapsis efficiency, as the number of phosphate\ngroups present at the DNA ends has been reported to influence synapsis (see PMID: 27924007).\n3. In the Discussion (lines 350–352), the authors mention the processing of incompatible sequences by Artemis. They\nshould provide a more detailed explanation of how Artemis processes these sequences and how this activity relates to\nNHEJ synapsis.\n4. The authors should include statistical analyses for all quantitative plots, specifying the number of single molecules\nanalyzed and the number of experimental replicates performed.\nMinor issues:\n5. In Figures S2f and S3a, the authors should include schematic diagrams illustrating the configurations of the positive-\ncontrol substrates to improve clarity and interpretability.\n6. The manner of reporting data (e.g., as number of molecules vs. number of events) is inconsistent across figure legends.\nThe authors should adopt a unified presentation or clearly justify the variation.\n7. Several typographical errors are present in the manuscript (e.g., “EFRET” in the Figure 3 legend). To improve accessibility\nfor a broad readership, please include a brief introduction to XLF (e.g., its full name and primary function in NHEJ) when it is\nfirst mentioned in the main text. The authors should perform a thorough proofreading to ensure accuracy and consistency\nthroughout the text.\n8. The experimental setup for the smFRET measurements should be described in greater detail. Please specify the key\nmicroscope configurations used, including the objective lens, laser wavelengths, emission filters, and whether temperature\nwas controlled during the imaging process.", "reviewer_3": "This manuscript by Zhang et al. presents a meticulous mechanistic study that fundamentally advances our understanding of\nnon-homologous end joining (NHEJ). By employing single-molecule FRET with a series of well-designed DNA substrates,\nthe authors systematically dissect how DNA end configurations regulate the formation and architecture of synaptic\ncomplexes. The work yields several key and novel findings: first, it conclusively demonstrates that the presence of\nmicrohomology (≥3 nt) at DNA ends can efficiently drive the formation of stable, ligation-competent close synaptic (CS)\ncomplexes through distinct structural pathways, even in the absence of the critical facilitator XLF. Second, it establishes a\nclear hierarchy where end complementarity can functionally substitute for XLF, thereby defining a microhomology-\ndependent bypass pathway for NHEJ synapsis. Third, it reveals that DNA end chemistry, specifically 5' phosphorylation,\nacts cooperatively with base pairing to promote synapsis. Overall, this study provides a new paradigm for understanding the\nflexibility and substrate-driven nature of the NHEJ machinery. I recommend publication after addressing the comments\noutlined below.\nMajor Comments\n1. In Figure 2, the assignment of the four distinct FRET states to specific synaptic complex conformations is central to the\nmanuscript's conclusions. While references are made to pre-ligated controls, the logical flow from the control FRET values to\nthe assignment of states like the \"junction-contacting\" (E4) and \"end-pairing\" (E3) complexes in the experimental data could\nbe more explicitly detailed in the main text or figure legends for the general reader.\n2. The claim that observed FRET signals represent protein-mediated synapsis requires solidification by including negative\ncontrol data. Quantification of the background frequency of co-localized Cy3/Cy5 spots (i.e., apparent FRET pairs) in\nexperiments performed with DNA substrates alone (in the absence of all proteins) should be provided in the supplement.\nThis is essential to rule out the contribution of any rare, non-specific DNA-DNA interactions to the reported synapsis events.\n3. The ensemble ligation assay in Fig. 1a uses 5' P-terminated DNA, whereas the smFRET assays in Figs. 1-4 use 5' OH-\nterminated DNA to isolate synapsis. Given that 5' P is itself a synapsis-promoting factor (Fig. 5), this discrepancy should be\nexplicitly acknowledged.\n4. The study focuses exclusively on perfectly complementary overhangs. To better contextualize the findings within the\nphysiological reality of diverse DSB ends, the authors should discuss the potential implications of their model for partially\ncomplementary or non-complementary ends. For instance, how would a 4-nt overhang with only 2-nt of microhomology\nbehave? Would it exhibit dynamic flickering, and how would XLF influence this?\n5. The primary overhangs used are G/C-rich, with ACTG/CAGT as a control. To generalize the conclusion that the effect\ndepends on \"complementarity rather than base composition,\" it would be strengthening to include data or a discussion\naddressing whether A/T-rich overhangs of equivalent length exhibit similar synapsis efficiency and CS complex stability.\n6. The data clearly show that XLF is dispensable for CS formation with overhangs ≥3 nt. However, the manuscript would\nbenefit from a deeper discussion on the potential role(s) of XLF under these conditions.\nMinor Comments\n1. There appears to be a discrepancy in the relative ligation efficiency of blunt ends versus overhangs when comparing Fig.\n1a and Fig. S6. Additionally, in Fig. S6, what explains the doublet band for the GGG/CCC substrate, and why might the G/C\noverhang exhibit lower efficiency than the blunt end.\n2. The legend for Fig. 4f notes that data for the critical GGG/CCC condition (which defines the 3-nt threshold) are from\nduplicate experiments (n=2). A sample size of n=2 is insufficient for reliable calculation of standard deviation. These data\nshould be supported by at least n=3 biological replicates to uphold this central claim.\n3. In FRET histograms (e.g., Figs. 2b, 3f-h, 4b-e), the colored Gaussian fits corresponding to different states (E1-E4, FS, CS)\nare not explicitly defined in the legends. Providing a clear color-to-state mapping directly in the figure legends would\nsignificantly improve readability and data interpretation.\n4. Page 5, line 135-136, The phrasing \"with the XLF present\" seems awkward and grammatically imprecise. For better\nclarity and formal academic style, it would be preferable to use a standard expression such as \"in the presence of XLF\" or\n\"when XLF is present\"."}, "author_response": "Responses to Reviewer Comments (author responses in BLUE)\nWe sincerely thank the Reviewers and the Editor for their time and constructive suggestions. We\nhave carefully revised the manuscript and believe that all comments have been fully addressed.\nReviewer #1\nThis is a review of NCOMMS-25-67092. In this study, the authors use a previously described\nbiophysics approach for measuring single molecule DNA synapsis by core factors in the non-\nhomologous end joining pathway. The experiments are straightforward with appropriate controls. The\nmain finding is that adding compatible DNA overhangs enhances synapsis via KU/XRCC4/LIG4 and\nreduces the requirement for XLF. My concern is that the relative requirements for NHEJ factors for\ndistinct end configurations have been relatively well established, and as such these findings are not\nvery surprising. One example is this paper from 2016 (PMID: 27703001). Much of the research on\nend synapsis, including from this group, focuses on blunt DNA ends merely because that end\nconfiguration needs to be stabilized without annealing. Other studies have also addressed the impact\nof non-homologous tails too. The authors mention that other end configurations are likely found in\ncells, such as damaged/oxidized bases, but such end configurations were not tested here, but rather\nfocus on microhomology substrates. Perhaps some experiments with other such substrates would\nimprove the scope of the study.\nWe appreciate the Reviewer’s thoughtful comments regarding the novelty of our work. It is true that\nprevious studies have examined how distinct end configurations influence NHEJ factor requirements\nand the repair (e.g., PMID: 27703001, PMID: 27924007, PMID: 28930678, and PMID: 24989324),\nand our Introduction also acknowledges these contributions (Page 4). However, despite extensive\nbiochemical characterizations, how different DNA ends determine synapsis architecture and\nfactor dependence at a mechanistic and structural level has remained unclear. As Reviewer 3\nalso noted, our study provides the first systematic single-molecule dissection of how end\nconfigurations govern the formation, stability, and architecture of NHEJ synaptic complexes.\nImportantly, the synapsis model that emerges from our work has direct implications for\nunderstanding how NHEJ synapsis coordinates with Artemis-mediated end processing—a major\nfocus of ongoing research in our laboratory.\nAs suggested, we have now tested overhangs containing one or two 8-oxoguanine(s) located\nat different positions. We find that a single 8-oxoguanine in the 4-nt 3’ overhang does not impair\nsynapsis efficiency, whereas two 8-oxoguanines produce a modest reduction in synapsis mediated\nby Ku and X4L4 (Fig. S3c). Additionally, neither one nor two 8-oxoguanines disrupt the formation or\nstability of CS complexes compared with undamaged GGGG/CCCC overhangs (Fig. S3d–S3f). These\ndata indicate that NHEJ can tolerate 8-oxoguanine lesions, consistent with previous reports (PMID:\n28930678 and PMID: 24989324). Lesions retained after NHEJ may subsequently be repaired by other\npathways.\nFinally, the mechanistic insight gained here into how such changes (and others discussed in\nthe manuscript) destabilize or modulate synapsis is essential for understanding the extent to which\nthe synapsis step does—or does not—restrict progression to the final ligation stage. In our view, these\nadvances underscore why our study merits publication in Nature Communications.\ncontaining 8-oxoguanine.\na, Schematic of the smFRET assay used to examine synapsis of GGGG/CCCC overhangs containing\n8-oxoguanine (8-oxoG) at defined positions. The 8-oxoG within the 3′ GGGG overhang is highlighted\nin red.\nb, Normalized synapsis efficiency of GGGG/CCCC substrates containing 8-oxoG in the presence or\nabsence of Ku plus X4L4. Data are presented as mean ± SD from at least three independent replicates.\nT-test (unpaired, two-tailed) was applied for p value calculation. *** : p < 0.001.\nc, Normalized synapsis efficiency of GGGG/CCCC substrates with or without 8-oxoG in reactions\nmediated by Ku plus X4L4. Data are presented as mean ± SD from at least three independent\nreplicates. T-test (unpaired, two-tailed) was applied for p value calculation. ns : p > 0.05, *** : p <\n0.001.\n2\nd, Histograms of E values for all synaptic complexes formed with GGGG/CCCC substrates\nFRET\ncontaining 8-oxoG. The E values shown correspond to the peak centers obtained from Gaussian fits.\nEvent numbers: N = 408 events, N = 213 events, N\n[GGG(8-oxoG)/CCCC] [(8-oxoG)GGG/CCCC] [(8-oxoG)G(8-oxoG)G/CCCC]\n= 181 events.\ne, Fractions of FS and CS complexes formed on GGGG/CCCC overhangs with or without 8-oxoG in\nthe presence of Ku and X4L4. Data are presented as mean ± SD from at least three independent\nreplicates. T-test (unpaired, two-tailed) was applied for p value calculation. ns : p > 0.05, * : p < 0.05,\n*** : p < 0.001.\nf, Dwell times of CS and FS complexes for GGGG/CCCC overhangs with or without 8-oxoG mediated\nby Ku and X4L4. Data are presented as mean ± SD from at least three independent replicates. T-test\n(unpaired, two-tailed) was applied for p value calculation. ns : p > 0.05.\nNote: In the figure, the 8-oxoG incorporated within the 3′ GGGG overhang is highlighted in red.\nReviewer #2\nIn this manuscript, Zhang et al. present an elegant and well-executed study investigating the role of\nDNA end configurations in NHEJ-mediated synapsis. Using a combination of biochemical and single-\nmolecule FRET analyses, the authors convincingly demonstrate that DNA end configurations—\nincluding terminal sequences and 5′ phosphate groups—play critical roles in determining the sub-\npathway choice of NHEJ synapsis. Given that broken DNA ends generated during chemo-\n/radiotherapy, CRISPR-Cas9-mediated gene editing, V(D)J recombination, and class-switch\nrecombination exhibit diverse end structures, understanding how these configurations influence NHEJ\nsynapsis has significant implications for genome stability, gene editing, and antibody maturation. This\nmakes the work highly relevant to the readership of Nature Communications. I am generally supportive\nof this study, but several concerns should be addressed before acceptance.\nWe thank the Reviewer for the positive assessment of our work and for the helpful suggestions that\nhave strengthened the manuscript.\nMajor concerns:\n1. The authors should provide gel images or Western blots to demonstrate the quality of the\nrecombinant Ku70–Ku80, XRCC4–LIG4, and XLF proteins. Additionally, activity assays of the purified\nproteins should be included, as the purity and functional integrity of these proteins are crucial for\nsupporting the conclusions drawn in this study.\nWe thank the Reviewer for this important point. SDS–PAGE analysis of all purified proteins is now\nincluded in Fig. S1a and shows >90% purity. Ligation activity assays for X4L4 are provided in both\nFig. 1a and Fig. S1k (previously Fig. S1j). As shown, X4L4 efficiently ligates compatible overhangs,\nKu stimulates ligation, and XLF robustly enhances blunt-end ligation. Together, these results confirm\nthat all recombinant proteins used in our study are fully active.\nFigure S1a. Purified human Ku70-Ku80 (Ku), XRCC4-Ligase IV (X4L4), and XLF proteins.\n3\n2. In Figure 5, the authors examine the effects of 5′ P on NHEJ synapsis using duplexes with 5′\nphosphorylated ends on both strands. The authors should also test the effect of a single 5′ P group\non synapsis efficiency, as the number of phosphate groups present at the DNA ends has been\nreported to influence synapsis (see PMID: 27924007).\nWe appreciate this insightful suggestion. We performed additional experiments (Figs. S7c – S7h) and\nupdated the corresponding text (Lines 339–342). Our results indicate that a single 5’ phosphate\npromotes CS formation by Ku + X4L4 to a similar extent as two phosphorylated ends, although the\ndual-phosphate configuration provides a modest additional enhancement for GG/CC overhangs (Figs.\n5e and S7h). These findings reinforce that the 5’ phosphate acts as a critical recognition element for\nLigase IV adenylation and thereby contributes to efficient synapsis.\nFigure S7c – S7h.\nc, Schematic of the smFRET assay for synapsis of an 85-bp phosphorylated blunt or 3′ overhang with\na 74-bp solution dsDNA bearing 5′-OH blunt or 3′ overhang.\nd-g, Histograms of E values for synaptic complexes formed using duplexes containing a single 5’\nFRET\nphosphate group on only one duplex end, mediated by Ku and X4L4. The E value shown was obtained\nfrom a Gaussian fit of the highest peak of the synaptic complexes. d, N = 31 events; e, N = 62 events;\nf, N = 125 events; g, N = 93 events.\nh, Fractions of FS and CS complexes formed by Ku and X4L4 with duplexes containing a single 5’\nphosphate group on only one duplex. Data are represented as mean ± SD of at least three\nindependent replicates.\n3. In the Discussion (lines 350–352), the authors mention the processing of incompatible sequences\nby Artemis. They should provide a more detailed explanation of how Artemis processes these\nsequences and how this activity relates to NHEJ synapsis.\nWe thank the Reviewer for the helpful suggestion. We have expanded the Discussion with a clearer\nexplanation of Artemis function (Lines 388–393, Page 13):\n4\n“Artemis, through its endonuclease activity, cleaves incompatible flaps near ssDNA–dsDNA junctions\nwithin the synaptic complexes. The resulting compatible overhangs can then efficiently promote and\nstabilize CS (Figs. 3 and S5) and ultimately enable end ligation by Ku and X4L4.”\n4. The authors should include statistical analyses for all quantitative plots, specifying the number of\nsingle molecules analyzed and the number of experimental replicates performed.\nThank you for the very helpful suggestions regarding the statistical reporting of our single-molecule\ndata. We have now included P-values, the number of analyzed single molecules, and the number of\nexperimental replicates for all smFRET measurements in both the main and supplemental figure\nlegends.\nMinor issues:\n5. In Figures S2f and S3a, the authors should include schematic diagrams illustrating the\nconfigurations of the positive-control substrates to improve clarity and interpretability.\nWe thank the Reviewer for the suggestion. Schematic diagrams illustrating the configurations of\npositive-control substrates have been added to Figs. S2h (previously S2f) and S4a (previously S3a).\n6. The manner of reporting data (e.g., as number of molecules vs. number of events) is inconsistent\nacross figure legends. The authors should adopt a unified presentation or clearly justify the variation.\nWe thank the Reviewer for pointing out this inconsistency. We have now standardized all reporting to\nthe number of events throughout the revised manuscript.\n7. Several typographical errors are present in the manuscript (e.g., “EFRET” in the Figure 3 legend).\nTo improve accessibility for a broad readership, please include a brief introduction to XLF (e.g., its full\nname and primary function in NHEJ) when it is first mentioned in the main text. The authors should\nperform a thorough proofreading to ensure accuracy and consistency throughout the text.\nWe thank the Reviewer for pointing out the typographical errors. We have thoroughly reviewed the\nmanuscript and corrected all typos. We have also introduced XLF with its full name and function in\nNHEJ at its first mention (Line 78, Page 3).\n8. The experimental setup for the smFRET measurements should be described in greater detail.\nPlease specify the key microscope configurations used, including the objective lens, laser\nwavelengths, emission filters, and whether temperature was controlled during the imaging process.\nWe thank the Reviewer for the suggestion about the details of our microscope system. We have added\na detailed description of the single-molecule imaging configuration (objective, laser wavelengths,\nemission filters, etc.) to the Methods section (Page 17). Reactions were performed at room\ntemperature without explicit temperature control (Line 523, Page 18).\nReviewer #3\nThis manuscript by Zhang et al. presents a meticulous mechanistic study that fundamentally advances\nour understanding of non-homologous end joining (NHEJ). By employing single-molecule FRET with\na series of well-designed DNA substrates, the authors systematically dissect how DNA end\nconfigurations regulate the formation and architecture of synaptic complexes. The work yields several\nkey and novel findings: first, it conclusively demonstrates that the presence of microhomology (≥3 nt)\nat DNA ends can efficiently drive the formation of stable, ligation-competent close synaptic (CS)\ncomplexes through distinct structural pathways, even in the absence of the critical facilitator XLF.\nSecond, it establishes a clear hierarchy where end complementarity can functionally substitute for\nreveals that DNA end chemistry, specifically 5' phosphorylation, acts cooperatively with base pairing\nto promote synapsis. Overall, this study provides a new paradigm for understanding the flexibility and\nsubstrate-driven nature of the NHEJ machinery. I recommend publication after addressing the\ncomments outlined below.\nWe sincerely thank the Reviewer for the enthusiastic assessment and constructive comments.\nMajor Comments\n1. In Figure 2, the assignment of the four distinct FRET states to specific synaptic complex\nconformations is central to the manuscript's conclusions. While references are made to pre-ligated\ncontrols, the logical flow from the control FRET values to the assignment of states like the \"junction-\ncontacting\" (E4) and \"end-pairing\" (E3) complexes in the experimental data could be more explicitly\ndetailed in the main text or figure legends for the general reader.\nWe thank the Reviewer for the valuable suggestions. We have clarified the logic behind FRET-state\nassignments by expanding the main text (Lines 191–196, Page 7):\n“Because the Cy3 and Cy5 dyes are positioned 4 bp from the ssDNA–dsDNA junctions on all 3’\noverhang substrates, a direct in-line junction–junction contact between the 3’ GGGG/CCCC\noverhangs results in an 8-bp Cy3–Cy5 separation (Fig. 2d, E ). Full pairing of the GGGG and CCCC\n4\noverhangs produces a 12-bp separation with two nicks at the junctions (Fig. 2d, E ), whereas partial\n3\nin-line pairing generates a 13–15-bp separation with two gaps between the overhangs (Fig. 2d, E ).”\n3\n2. The claim that observed FRET signals represent protein-mediated synapsis requires solidification\nby including negative control data. Quantification of the background frequency of co-localized\nCy3/Cy5 spots (i.e., apparent FRET pairs) in experiments performed with DNA substrates alone (in\nthe absence of all proteins) should be provided in the supplement. This is essential to rule out the\ncontribution of any rare, non-specific DNA-DNA interactions to the reported synapsis events.\nWe understand the Reviewer’s concern regarding potential non-specific DNA–DNA interactions and\nappreciate the suggestion to include negative control data. Our previous work (PMID: 31399561)\ndemonstrated negligible synapsis for blunt-ended substrates in the absence of NHEJ proteins,\nindicating that the dsDNA regions do not generate background synapsis. The only possible source of\nnon-specific synapsis would be spontaneous pairing of compatible overhangs. To exclude this\npossibility, we performed DNA-only control experiments using the 3’ GGGG/CCCC overhang\nsubstrates, which have the highest intrinsic pairing stability among the overhangs tested. The results\nshow that background synapsis for these substrates is negligible (Fig. S1I).\nefficiency for 3′ GGGG/CCCC overhangs in the presence or absence of Ku plus X4L4. Substrates\nwith or without 5′ phosphate groups were tested. Data are presented as mean ± SD from at least\nthree independent replicates. T-test (unpaired, two-tailed) was applied for p value calculation. *** : p\n< 0.001.\n3. The ensemble ligation assay in Fig. 1a uses 5' P-terminated DNA, whereas the smFRET assays in\nFigs. 1-4 use 5' OH-terminated DNA to isolate synapsis. Given that 5' P is itself a synapsis-promoting\nfactor (Fig. 5), this discrepancy should be explicitly acknowledged.\nWe understand the Reviewer’s concerns regarding this discrepancy. Because a 5’ phosphate is\nessential for X4L4–mediated ligation, the ensemble ligation experiment in Fig. 1a uses 5’-\nphosphorylated substrates to illustrate that end overhangs contribute to forming ligation-competent\nCS complexes—a prerequisite for ligation. In contrast, the substrates used in Figs. 1–4 carry 5’ OH\ngroups to ensure that we measure only synapsis, independent of ligation, since 5’ OH ends cannot\nbe ligated by X4L4. We have added the following sentence to clarify this rationale (Lines 148-149,\nPage 6): “Both substrates carry 5’ OH ends, allowing us to focus exclusively on synapsis rather than\nligation.”\n4. The study focuses exclusively on perfectly complementary overhangs. To better contextualize the\nfindings within the physiological reality of diverse DSB ends, the authors should discuss the potential\nimplications of their model for partially complementary or non-complementary ends. For instance, how\nwould a 4-nt overhang with only 2-nt of microhomology behave? Would it exhibit dynamic flickering,\nand how would XLF influence this?\nThank you very much for the valuable suggestions. As part of our ongoing work on Artemis-coupled\nsynapsis, we now discuss the mechanistic implications for (partially) incompatible overhangs and\nhave added new data on 3-nt microhomology (GGGTT/CCCTT) substrates (Fig. S5). We designed\nGGGTT/CCCTT overhangs, rather than the suggested GGTT/CCTT, because 3-nt microhomology is\nrequired for CS formation by Ku plus X4L4 in the absence of 5’ P. Our results show that Ku plus X4L4\ncan promote CS formation with GGGTT/CCCTT overhangs; however, the stability and formation\nefficiency of the CS complex are lower than those observed for GGG/CCC overhangs (dwell: 4.2 s vs.\n10.3 s; fraction: 19.6% vs. 34.8%) (Figs. 3i, S4c, and S5e – S5f). XLF substantially enhances both\nformation and stabilization of CS complexes for the GGGTT/CCCTT overhangs (Figs. S5d and S5g).\nThese results indicate that incompatible overhangs can reduce synapsis by Ku plus X4L4, while XLF\nplays a critical role in mediating synapsis of partially compatible overhangs. Formation of the CS\ncomplex with partially compatible overhangs generate incompatible flaps for Artemis-mediated\nprocessing, which may further promote CS formation. We have now discussed these findings in detail\nin the revised text.\nX4L4, with further enhancement in the presence of XLF.\na, Schematic of the smFRET assay used to examine synapsis of GGGTT/CCCTT overhangs\nmediated by Ku plus X4L4 with or without XLF.\nb, Normalized synapsis efficiency of 3’ GGGTT/CCCTT overhangs mediated by Ku, X4L4, and XLF.\nData are presented as mean ± SD from at least three independent replicates. T-test (unpaired, two-\ntailed) was applied for p value calculation. * : p < 0.05, *** : p < 0.001.\nc-d, Histograms of E values for all the synaptic complexes formed with 3′ GGGTT/CCCTT\nFRET\noverhangs mediated by Ku plus X4L4 in the absence (c) or presence (d) of XLF. The E value shown\nwas obtained from a Gaussian fit of the highest peak of the synaptic complexes. c, N = 182 events;\nd, N = 562 events.\ne, Fractions of FS and CS complexes formed on GGGTT/CCCTT overhangs mediated by Ku, X4L4\nand XLF. Data are presented as mean ± SD from at least three independent replicates. T-test\n(unpaired, two-tailed) was applied for p value calculation. *** : p < 0.001.\n8\nf-g, Histogram and corresponding exponential fits of synapsis dwell times for CS events mediated by\nKu plus X4L4 in the absence (f, N = 40 events) or presence (g, N = 68 events) of XLF. Only the events\nwith both start and end time points within the detection time window were included.\n5. The primary overhangs used are G/C-rich, with ACTG/CAGT as a control. To generalize the\nconclusion that the effect depends on \"complementarity rather than base composition,\" it would be\nstrengthening to include data or a discussion addressing whether A/T-rich overhangs of equivalent\nlength exhibit similar synapsis efficiency and CS complex stability.\nWe appreciate the Reviewer’s concerns regarding the effects of base composition on CS formation.\nBased on our observations of CS formation and the FS-to-CS transition mediated by Ku plus X4L4\nfor both GGGG/CCCC and ACTG/CAGT overhangs, we concluded in the initial submission that Ku\nplus X4L4 promote CS formation primarily depending on overhang compatibility rather than base\ncomposition. Here, we focus on CS formation independent of base composition. However, we agree\nwith the Reviewer that base composition can influence the efficiency of CS formation. To address this,\nwe tested CS formation for TTTT/AAAA overhangs as suggested. The results show that CS can still\nform, but with relatively lower efficiency (Figs. 2b and S2e - S2g), likely because T/A-rich overhangs\nhave lower pairing stability compared to other compatible overhangs. These results reinforce that\ncomplementarity, rather than base composition, is the primary determinant of synapsis, although base\ncomposition can modulate its efficiency.\nFigure S2f - S2g.\nf, Histograms of E values of all the synaptic complexes formed with the duplexes with 3’\nFRET\nTTTT/AAAA overhangs. The E values shown were obtained from the Gaussian fits of the\ncorresponding peaks of the synaptic complexes. N= 240 events.\ng, Normalized synapsis efficiency for 3’ TTTT/AAAA overhangs mediated by Ku and X4L4. T-test\n(unpaired, two-tailed) was applied for p value calculation. *** : p < 0.001.\n6. The data clearly show that XLF is dispensable for CS formation with overhangs ≥3 nt. However,\nthe manuscript would benefit from a deeper discussion on the potential role(s) of XLF under these\nconditions.\nWe appreciate your valuable suggestions. As noted in point 4, our new results show that XLF\npromotes CS formation for partially compatible overhangs (≥3-nt microhomology). This process\ngenerates incompatible flaps that can be processed by Artemis, thereby facilitating NHEJ ligation.\nWe have incorporated this discussion in the revised manuscript (Lines 388-393, Page 13).\nMinor Comments\n9\n1. There appears to be a discrepancy in the relative ligation efficiency of blunt ends versus overhangs\nwhen comparing Fig. 1a and Fig. S6. Additionally, in Fig. S6, what explains the doublet band for the\nGGG/CCC substrate, and why might the G/C overhang exhibit lower efficiency than the blunt end.\nWe apologize for the confusion. The ligation efficiencies for blunt ends are consistent (8.1% in Fig. 1a\nvs. ~9% in Fig. S8). The label for overhangs in Fig. 1a has now been clarified as GGGG/CCCC.\nThe doublet band observed for the GGG/CCC substrate arises from excess unpaired Cy5-\nlabeled ssDNA, which does not affect X4L4-mediated ligation. A note regarding this has been added\nto the figure legend.\nRegarding the lower ligation efficiency of G/C overhangs compared to blunt ends, we repeated\nthe experiments multiple times with consistent results. Although the underlying mechanism remains\nunclear, these ligation efficiencies are in agreement with our synapsis data. Specifically, G/C\noverhangs form fewer and less stable CS complexes than blunt-ended overhangs when mediated by\nKu+X4L4+XLF (Figs. 4f, 4g), which corresponds to their lower ligation efficiency (Fig. S8).\n2. The legend for Fig. 4f notes that data for the critical GGG/CCC condition (which defines the 3-nt\nthreshold) are from duplicate experiments (n=2). A sample size of n=2 is insufficient for reliable\ncalculation of standard deviation. These data should be supported by at least n=3 biological replicates\nto uphold this central claim.\nWe appreciate your suggestion. We have performed additional biological replicates (now n ≥ 3).\n3. In FRET histograms (e.g., Figs. 2b, 3f-h, 4b-e), the colored Gaussian fits corresponding to different\nstates (E1-E4, FS, CS) are not explicitly defined in the legends. Providing a clear color-to-state\nmapping directly in the figure legends would significantly improve readability and data interpretation.\nThank you for the helpful suggestions. We have updated the figure legends of Figures S1e and S1f\nto explicitly define the color-to-state mapping (FS and CS). For the E –E states, we have directly\n1 4\nlabeled each state on the corresponding fitted peaks.\n4. Page 5, line 135-136, The phrasing \"with the XLF present\" seems awkward and grammatically\nimprecise. For better clarity and formal academic style, it would be preferable to use a standard\nexpression such as \"in the presence of XLF\" or \"when XLF is present\".\nThank you very much for pointing out this grammatic error. We have corrected this statement to “in\nthe presence of XLF.”\nResponses to Reviewer Comments (author responses in BLUE)\nWe sincerely thank the Reviewers and the Editor for their time and support of our study.\nReviewer #1\nThe revised manuscript contains new data in Figures S5 and S6, which include data with 8-oxoG, and\nhence extend their analysis to additional end configurations. As such, my concerns have been\nadequately addressed. My remaining suggestion is that perhaps Figs S5 and S6 could be moved to\nthe main manuscript, should space allow.\nWe thank the Reviewer for the suggestion to move some of the supplementary data into the main\nmanuscript. In the revised version, Figure S5 presents synapsis of substrates containing embedded\nmicrohomologies, while Figure S6 shows representative ROI images of synapsis for G/C, GG/CC,\nGGG/CCC, and GGGG/CCCC overhangs. Figure S6 was included in the initial submission and does\nnot involve experiments with 8-oxoG. We therefore believe that the Reviewer was referring to Figure\nS3 in the revised manuscript, which contains the data related to 8-oxoG.\nAccordingly, as suggested by the Reviewer, we have moved the main results from Figures S3\nand S5 into the main manuscript as follows:\n1. Previous Figures S3a, S3c, S3d, S3e, and S3f were incorporated into new Figure 2 as\nFigures 2e, 2f, 2g, 2h, and 2i, respectively. Previous Figures S3a and S3b remain in the new Figure\nS3.\n2. Previous Figures S5a, S5c, S5e (Ku+X4L4 condition), and S5f were incorporated into new\nFigure 3 as Figures 3k, 3l, 3m, and 3n, respectively.\n3. Previous Figures S5d, S5e (Ku+X4L4+XLF condition), and S5g were incorporated into new\nFigure 4 as Figures 4h, 4i, and 4j, respectively. Previous Figures S5a and S5b remain in the new\nFigure S5.\nReviewer #2\nI have carefully reviewed the authors' responses and the accompanying revisions to the manuscript\nand supplementary materials. The revisions have substantially improved the manuscript. I am now\nfully satisfied with the manuscript and believe it meets the high standards for publication in Nature\nCommunications.\nWe sincerely thank the Reviewer for recommending our manuscript.\nReviewer #3\nI read the revised version of this manuscript with great interest and was particularly pleased to see\nthat the authors have thoughtfully addressed the various comments raised during the initial review\nprocess. The responses provided are convincing. Overall, I am satisfied with the revisions and now\nsupport the acceptance of this manuscript in Nature\nCommunications.\nWe sincerely thank the Reviewer for recommending our manuscript.\n1"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised manuscript contains new data in Figures S5 and S6, which include data with 8-oxoG, and hence extend their analysis to additional end configurations. As such, my concerns have been adequately addressed. My remaining suggestion is that perhaps Figs S5 and S6 could be moved to the main manuscript, should space allow.", "reviewer_2": "I have carefully reviewed the authors' responses and the accompanying revisions to the manuscript and supplementary materials. The revisions have substantially improved the manuscript. I am now fully satisfied with the manuscript and believe it meets the high standards for publication in Nature Communications.", "reviewer_3": "I read the revised version of this manuscript with great interest and was particularly pleased to see that the authors have thoughtfully addressed the various comments raised during the initial review process. The responses provided are convincing. Overall, I am satisfied with the revisions and now support the acceptance of this manuscript in Nature Communications."}, "author_response": "NCOMMS-25-67092B\nResponses to Reviewer Comments (author responses in BLUE)\nWe sincerely thank the Reviewers and the Editor for their time and support of our study.\nReviewer #1\nThe revised manuscript contains new data in Figures S5 and S6, which include data with 8-oxoG, and\nhence extend their analysis to additional end configurations. As such, my concerns have been\nadequately addressed. My remaining suggestion is that perhaps Figs S5 and S6 could be moved to\nthe main manuscript, should space allow.\nWe thank the Reviewer for the suggestion to move some of the supplementary data into the main\nmanuscript. In the revised version, Figure S5 presents synapsis of substrates containing embedded\nmicrohomologies, while Figure S6 shows representative ROI images of synapsis for G/C, GG/CC,\nGGG/CCC, and GGGG/CCCC overhangs. Figure S6 was included in the initial submission and does\nnot involve experiments with 8-oxoG. We therefore believe that the Reviewer was referring to Figure\nS3 in the revised manuscript, which contains the data related to 8-oxoG.\nAccordingly, as suggested by the Reviewer, we have moved the main results from Figures S3\nand S5 into the main manuscript as follows:\n1. Previous Figures S3a, S3c, S3d, S3e, and S3f were incorporated into new Figure 2 as\nFigures 2e, 2f, 2g, 2h, and 2i, respectively. Previous Figures S3a and S3b remain in the new Figure\nS3.\n2. Previous Figures S5a, S5c, S5e (Ku+X4L4 condition), and S5f were incorporated into new\nFigure 3 as Figures 3k, 3l, 3m, and 3n, respectively.\n3. Previous Figures S5d, S5e (Ku+X4L4+XLF condition), and S5g were incorporated into new\nFigure 4 as Figures 4h, 4i, and 4j, respectively. Previous Figures S5a and S5b remain in the new\nFigure S5.\nReviewer #2\nI have carefully reviewed the authors' responses and the accompanying revisions to the manuscript\nand supplementary materials. The revisions have substantially improved the manuscript. I am now\nfully satisfied with the manuscript and believe it meets the high standards for publication in Nature\nCommunications.\nWe sincerely thank the Reviewer for recommending our manuscript.\nReviewer #3\nI read the revised version of this manuscript with great interest and was particularly pleased to see\nthat the authors have thoughtfully addressed the various comments raised during the initial review\nprocess. The responses provided are convincing. Overall, I am satisfied with the revisions and now\nsupport the acceptance of this manuscript in Nature\nCommunications.\nWe sincerely thank the Reviewer for recommending our manuscript.\n1"}]

Decision letter