{"paper_id":"e1bf4691-c214-43a3-b9ea-d021548669aa","body_text":"1 \nDNA polymerase Lambda is anchored within the NHEJ synaptic complex 1 \nvia Ku70/80 2 \n 3 \nPhilippe Frit 1†, Himani Amin 2†, Sayma Zahid 2†, Nadia Barboule 1†, Chloe Hall 2, Gurdip 4 \nMatharu2, Steven W. Hardwick 3, Jeanne Chauvat 1, Sébastien Britton 1, Dima Y. Chirgadze3, 5 \nVirginie Ropars4, Jean-Baptiste Charbonnier4, Patrick Calsou1*, Amanda K. Chaplin2‡*. 6 \n 7 \n1. Institut de Pharmacologie et Biologie Structurale (IPBS), Université de Toulouse, 8 \nCNRS, Université Toulouse III - Paul Sabatier (UT3), Toulouse, France. 9 \n2. Leicester Institute for Structural and Chemical Biology, Department of Molecular and 10 \nCell Biology, University of Leicester; Leicester, UK. 11 \n3. Department of Biochemistry, University of Cambridge; Sanger Building, Tennis Court 12 \nRoad, Cambridge, CB2 1GA, United Kingdom. 13 \n4. Institute for Integrative Biology of the Cell (I2BC), Institute Joliot, CEA, CNRS, 14 \nUniversité Paris-Saclay; 91198, Gif-sur-Yvette cedex, France 15 \n 16 \n†These authors contributed equally 17 \n*Corresponding authors. Email: Amanda Chaplin ac853@leicester.ac.uk and Patrick Calsou 18 \ncalsou@ipbs.fr 19 \n 20 \n‡Lead contact: Amanda Chaplin ac853@leicester.ac.uk 21 \n 22 \n 23 \n 24 \n 25 \n 26 \n 27 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 2 \nAbstract 28 \nNon-homologous end joining (NHEJ) is the predominant pathway by which double -strand 29 \nDNA breaks ( DSBs) are repaired in mammals. To enable final break closure, various NHEJ 30 \nend-processing factors respond to the chemistry of the damaged DNA ends . Amongst these 31 \nfactors is DNA polymerase lambda (Pol ), a member of the Pol X family. How members of 32 \nthe Pol X family engage with the NHEJ complex is unknown. Here, we present cryo -EM 33 \nstructures of Pol λ in complex with the Ku70/80 DSB sensor whilst engaged with the DNA -34 \nPK holoenzyme in a long-range synaptic complex. These structures reveal a specific interaction 35 \nsite between Ku70/80 and the Pol λ BRCT domain. The functionality of this interaction is 36 \nassessed by generating point mutations on either side of the Pol λ BRCT :Ku70/80 interface. 37 \nUsing these mutants in t wo orthogonal assays in cells (live protein recruitment at biphoton 38 \nlaser-damaged nuclear sites and transfection with an original gap-filling reporter plasmid) 39 \ndefines the molecular basis and essentiality of the BRCT domain for the recruitment and 40 \nactivity of the Pol λ within the NHEJ complex . Ultimately, these data explain the role of this 41 \ninteraction in cell survival to DSBs. Finally, we propose a unified model for the interaction of 42 \nthe three Pol X family members bearing BRCT domains with the same site of Ku70/80. 43 \n 44 \nIntroduction 45 \nNon-homologous end joining (NHEJ) is the predominant pathway in which double strand DNA 46 \n(dsDNA) breaks are repaired in mammals. Central to the process of NHEJ are large, multi -47 \nprotein complexes formed by the canonical proteins DNA -dependent protein kinase catalytic 48 \nsubunit (DNA-PKcs), the heterodimer of Ku70/80, DNA ligase IV (LigIV), X-ray repair cross-49 \ncomplementing protein 4 (XRCC4) and XRCC4 -like factor (XLF) 1. In addition, PAXX 50 \n(Paralog of XRCC4 and XLF) is a n accessory NHEJ protein identified more recently which 51 \nhas functional redundancy with XLF in the NHEJ mechanism 2,3. Although this repair pathway 52 \nis a complex process, it is generally considered to proceed via three major steps; DNA end 53 \nrecognition, DNA end processing and finally DNA ligation 4. 54 \nThe initial step of dsDNA break recognition relies predominantly on the Ku70/80 heterodimer, 55 \nwhich engages the DNA ends and subsequently recruits the DNA -PKcs kinase, together 56 \nforming the DNA-PK holoenzyme 5. Recent cryo-electron microscopy (cryo-EM) structures of 57 \nDNA-PK assemblies revealed how DNA substrates can be positioned for efficient synapsis via 58 \ndimerization of this large enzyme. Intriguingly, DNA-PK has two distinct mechanisms to form 59 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 3 \na pre-synaptic complex, corresponding to alternate structural dimers of DNA -PK. It was first 60 \nproposed that DNA-PK can synapse the broken DNA ends using a dimer of DNA-PK mediated 61 \nby the C -terminal region of Ku80 (herein termed Ku80 -mediated dimer ) 6. Soon after, an 62 \nalternative DNA-PK dimer complex was described, which can form upon addition of XRCC4, 63 \nLigIV and XLF 2,7,8. In this second complex, the DNA-PK dimer is crucially bridged by XLF 64 \n(herein termed XLF-mediated dimer). In both assemblies, the distance between the damaged 65 \nDNA ends is identical at 115 Å. This distance is not close enough for DNA ligation to proceed 66 \nwithout further rearrangement of the assembly , therefore both dimeric assemblies have been 67 \nreferred to as long-range synaptic complexes (LRC). We have shown recently that PAXX can 68 \nreplace XLF for bridging the Ku80-mediated DNA-PK dimer through binding to Ku70 but 69 \ndoes not replace XLF in the XLF mediated dimer 2. 70 \nThe second step in the NHEJ mechanism is end processing and is crucial if the DNA ends 71 \ncannot be readily ligated. Different end processing factors can be recruited to either trim or 72 \nextend the damaged ends via the action of DNA nucleases or polymerases, as detailed below. 73 \nThe final step of the repair mechanism involves ligation of the phosphodiester backbone with 74 \n5'-phosphate and 3' -hydroxyl moieties by LigIV, facilitated by XLF and XRCC4. To enable 75 \nligation, the LRC is thought to transition to a short -range complex (SRC) following 76 \nautophosphorylation and removal of DNA-PKcs 9. In this SRC, the damaged DNA ends are in 77 \nmuch closer proximity to each other and thereby are primed for DNA ligation. A cryo-EM 78 \nstructure of the SRC has been solved which shows the catalytic domain of LigIV interacting 79 \ndirectly with the DNA7. While structural studies have significantly improved understanding of 80 \nthe NHEJ mechanism by illuminating the architecture of several complexes central to the 81 \nprocess, how the end processing proteins, in particular polymerases, interact with these 82 \ncomplexes has remained an enigma. 83 \nThe end processing factors known to be implicated in NHEJ include nucleases such as Artemis 84 \nwhich can trim DNA nucleotides, and polymerases which can be used for end filling or 85 \nextension. Recent structural data has revealed how Artemis can engage with a DNA -PK 86 \nmonomer via its N -terminal nuclease domain and is positioned between the N -HEAT 87 \n(Huntingtin, Elongation Factor 3, A subunit of protein phosphatase 2A, Target of 88 \nRapamycin/TOR) and M -HEAT domain of DNA -PKcs 10. To date however, the structural 89 \nmechanism of interaction between DNA polymerases and the NHEJ machinery has not been 90 \ndefined. The polymerases involved in NHEJ belong to the DNA Polymerases X family and 91 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 4 \ncatalyse the addition of nucleotides at the 3’-OH end of DNA. In mammals, three polymerases, 92 \nnamely Pol λ, Pol μ and terminal deoxynucleotidyl transferase (TdT), account for the majority 93 \nof DNA synthesis during NHEJ, with the latter mainly involved in V(D)J recombination 11,12 94 \n13. These polymerases share a common domain organisation which consists of a breast cancer 95 \ngene 1 (BRCA1) C -terminal (BRCT) region at the N -terminus followed by a C -terminal 96 \ncatalytic domain (Figure S1A). The catalytic region facilitates the protein :DNA interaction, 97 \nthus enabling the function of the enzyme. The chemical nature of the DNA ends is a key 98 \ndeterminant of the polymerase activity, with each of the three enzymes having varying degrees 99 \nof template-dependency. Pol λ appears to display strong activity on DNA ends that have a 100 \npaired primer terminus 14. Furthermore, due to its accuracy, it has been proposed that Pol λ is 101 \ngiven priority for gap filling in most cell types 15. The BRCT domains are thought to direct the 102 \npolymerases to sites of DNA damage via interactions with complexes comprising DNA and 103 \nKu70/80, or larger assemblies including the NHEJ proteins DNA-PKcs, LigIV and XRCC4 104 \n16,17. While the structure of the BRCT and catalytic domain of these polymerases have been 105 \nsolved in isolation 16,18, how these domains engage with each other, and larger NHEJ 106 \ncomplexes has remained unclear. 107 \nIn order to elucidate how the polymerases interact with NHEJ machineries, we collected cryo-108 \nEM data of the Ku80 mediated dimer of DNA-PK in complex with Pol λ. From our structural 109 \ndata we identify the specific binding region of the BRCT domain of Pol λ with the bridge region 110 \nof Ku70/80 within DNA-PK complex. Using mutagenesis studies in cells on both Pol λ and 111 \nKu70/80, we identified key residues that are critical for Pol λ anchoring in NHEJ complexes, 112 \nefficient gap-filling activity and ultimately cell survival to DSBs. Finally, we propose that all 113 \nmembers of the Polymerase X family share a conserved recognition site on the Ku70/80 114 \nheterodimer. 115 \n 116 \nResults 117 \nCryo-EM structure of Pol λ bound to the Ku80-mediated DNA-PK dimer 118 \nIn order to understand how Pol λ interacts with the NHEJ Long Range Complex (LRC), cryo-119 \nEM data was collected on a sample containing DNA -PKcs, Ku70/80, DNA, XRCC4, LigIV, 120 \nPAXX and Pol λ to allow formation of the Ku80 -mediated dimer of DNA -PK. Following 121 \nextensive particle classification, a consensus map was obtained of the Ku80-mediated dimer of 122 \nDNA-PK. The particles comprising this initial map were further characterized to generate maps 123 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 5 \nof the Ku80 -mediated dimer with and without XRCC4 and DNA LigIV (thereafter termed 124 \nLX4) engaged (Figure 1, S2-4). At this stage, it was apparent that there were no large areas of 125 \nadditional density that could accommodate the full-length Pol λ protein, when compared to 126 \nprevious maps of this DNA -PK dimer. However, a small region of additional density was 127 \napparent at the bridge that defines the thinner part of the Ku70/80 ring region (Figure S5). To 128 \nfocus specifically on this region, particles were re-extracted in smaller boxes corresponding to 129 \nthe monomeric size of DNA-PK, and maps were generated of single protomers from within the 130 \ndimeric assemblies (herein termed half-dimers) with and without LX4 bound. 131 \nThe structure without LX4 bound has an overall resolution of 4.53 Å and the map with LX4 132 \nbound has an overall resolution of 4.26 Å (Figure S2-5). Within both of these half-dimer maps, 133 \nDNA-PK along with the PAXX Ku-binding motif (P-KBM) could be docked (Figure 1). The 134 \nPAXX P-KBM can be seen interacting with the von Willebrand-like (vWA) domain of Ku70, 135 \nas has been previously characterised 2. Additionally, XRCC4 and BRCT tandem repeats of 136 \nLigIV can be docked into the map with LX4 bound (Figure 1). As seen in our previous 137 \nstructures there is a central helix in DNA-PKcs which blocks the DNA ends, which is only 138 \npresent when LX4 is engaged. Additional density can be observed at the bridge formed by 139 \nKu70 and Ku80 in both maps, into which the NMR structure of the Pol λ BRCT domain (PDB: 140 \n2JW5) could be confidently docked (Figure 1 and S2-5). Although the full-length Pol λ was 141 \nused, only the BRCT domain could be modelled into the cryo-EM map. This suggests that the 142 \ncatalytic domain has no stable interaction with DNA with the LRC in the cryo-EM assemblies 143 \ndetermined. 144 \n 145 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 6 \n 146 \nFigure 1: Cryo-EM structures of DNA-PK, PAXX and Pol  with and without LX4. A) DNA-PK 147 \n+ PAXX + Pol  dimeric map with extra density for Pol  highlighted in a dashed pink circle, with two 148 \norientations of processed half dimer presented below. DNA -PKcs in grey, Ku70 in orange, Ku80 in 149 \ngreen, DNA in yellow, PAXX in cyan and Pol  in purple. B) DNA-PK + PAXX + Pol  + LX4 with 150 \nextra density for Pol  highlighted in a dashed pink circle, with two orientations of processed half dimer 151 \npresented below. DNA-PKcs in grey, Ku70 in orange, Ku80 in green, DNA in yellow, LigIV in red, 152 \nXRCC4 in dark blue, PAXX in cyan and Pol  in purple. Inset, zoom in of the Pol  extra density. 153 \n 154 \nMolecular basis of the Pol λ BRCT interaction with Ku70/80 155 \nThe Pol λ BRCT domain is positioned at the interface formed by Ku70 and Ku80, at the 156 \nperiphery of the DNA binding channel formed by the Ku70/80 heterodimer . An interaction 157 \nbetween Pol λ and Ku70/80-LigIV-XRCC4 has been previously suggested, with the residues 158 \nproposed to be involved in the interaction being situated in α1 helix of the BRCT domain 16. In 159 \nagreement with this previous study, our structure shows that Pol λ BRCT domain is positioned 160 \nto allow helix α1 to dock into a groove formed between Ku70 and Ku80 (Figure 2D). 161 \nSpecifically, the amino acid residues Arg57 and Leu60 of Pol  BRCT domain appear to 162 \nmediate the contact with Ku70/80, in agreement with previous data, which found that mutations 163 \nof these residues prevented Pol  interaction on DNA with Ku70/80 or Ku70/80 -LigIV-164 \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 7 \nXRCC416,17. With regards to Ku70/80, the specific interaction sites encompass residues 165 \nbetween 301 – 310 on Ku80 and residues 301 – 308 on Ku70. 166 \n 167 \nMutagenesis of Ku70/80 interaction residues on Pol λ 168 \nBased on our structural data, we probed how the Pol λ BRCT interaction with Ku70/80 impacts 169 \nthe response to DNA damage in cells. We first analysed the influence on recruitment of Pol λ 170 \nat laser-induced DNA damage sites. To establish to what extent Pol λ mobilisation to damaged 171 \nsites was Ku -dependent, we used in -house engineered U2OS cells that allow circumventing 172 \nthe lethality of Ku loss in human cells as previously described 2,19 (Figure 2 and S6A). Briefly, 173 \nendogenous Ku70 expression was first knocked -down via the constitutive expression of an 174 \nshRNA associated with cell rescue through expression of Ku70 tagged with a mini -auxin 175 \ninducible degron (mAID). Within a few hours upon addition of auxin (indole -3-acetic acid, 176 \nIAA), degradation of endogenous Ku70 occurs with concomitant Ku80 depletion due to the 177 \nknown reciprocal stabilization of both Ku subunits. Full-length Pol λ fused to GFP was rapidly 178 \nand substantially recruited to DNA damage sites in the presence of Ku. This recruitment of the 179 \nfull-length polymerase was only partly Ku-dependent since it substantially persisted under Ku 180 \ndepletion conditions (Figure 2B, – red line). This may indicate an ability of Pol λ to interact 181 \ndirectly with DNA at sites of damage, or may be related to Ku-independent repair functions of 182 \nPol λ outside NHEJ 20. 183 \nIn contrast to data obtained with full -length Pol λ, when the fusion was restricted to the N -184 \nterminal Pol λ BCRT domain ( Figure S1A), its recruitment to micro -irradiated areas was 185 \nmostly Ku-dependent since it was nearly abolished without Ku (Figure 2C). Guided by our 186 \nstructural data, we next probed the impact of specific Pol λ BRCT mutations on the recruitment 187 \nto DNA damage sites (Figure 2D). As shown in Figure 2E, all the mutants tested impaired 188 \nGFP-Pol λ BRCT accrual at laser -induced DNA damage sites to various extents, with 189 \nmutations at R57 or L60 positions being the most detrimental. 190 \nNotably, while GFP -Pol λ expression was nuclear with a nucleolar enrichment, the latter 191 \ndisappeared upon Ku depletion (Figure S6B). The Pol λ BRCT nucleolar enrichment was also 192 \nstrongly reduced with all BRCT mutants tested (Figure S6C). Since it is known that nuclear 193 \nKu is enriched in the nucleolus in the absence of DNA damage 21, this suggests that GFP-Pol λ 194 \nlocalisation to the nucleolus relies on its interaction with Ku , and that this localisation is 195 \ndisrupted by specific mutations within the BRCT domain of Pol λ. 196 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 8 \n 197 \nFigure 2. Impact of mutations in the BRCT domain of Pol  on its recruitment to DSBs by Ku. A) 198 \nPrinciple of the laser micro -irradiation experiment. U2OS cells engineered for auxin (IAA) -induced 199 \nKu70 knockdown, rescued or not with wild -type (WT) Ku70 and expressing either GFP -tagged full-200 \nlength Pol  or its BRCT domain, were micro-irradiated with an 800 nm multiphoton laser to generate 201 \nDSBs in subnuclear areas. 202 \nB) Left panel: representative images before and 20 s after irradiation (irradiated areas are indicated by 203 \nwhite arrows) of nuclei from U2OS expressing GFP -tagged full-length Pol  and depleted or not of 204 \nKu70 (±Ku). Right panel: quantification of fluorescence accumulation at laser -induced DNA damage 205 \nsites. Results are plotted as mean values of at least 20 nuclei ± SEM. 206 \nC) Same as in B) with U2OS expressing GFP-tagged Pol -BRCT. 207 \nD) Position of mutated residues in the BRCT domain of Pol  (purple) at the interface with Ku70/Ku80 208 \n(orange and green, respectively). 209 \nE) Quantification of fluorescence accumulation at laser -induced DNA damage sites in U2OS cells 210 \nexpressing GFP-tagged WT or mutated BRCT domain of Pol . Results are plotted as mean values of 211 \nat least 20 nuclei ± SEM. 212 \n 213 \n 214 \n 215 \n \n \n \n \n \n \n \n \n \n \n \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 9 \nMutagenesis of Ku70/80 residues 216 \nTo identify key residues of Ku70/80 residues at the Pol λ BRCT :Ku interface, we replaced 217 \nendogenous Ku subunits with mutated forms of either Ku70 or Ku80 as guided by our structural 218 \ndata (Figure 3A, S7A-C). We then monitored the impact on Pol λ BRCT recruitment following 219 \nDNA damage . To ensure that mutants of Ku70/80 did not influence Ku mobilisation at 220 \ndamaged sites per se, we monitored simultaneously the recruitment of mCherry -PAXX co-221 \nexpressed within the same cells, since PAXX recruitment is strictly Ku-dependent 2 (Figure 222 \n3B-F). Mutations on the positions F303, L310 on Ku70 or E292, E304 on Ku80 nearly 223 \nabolished Pol λ recruitment (Figure 3D, 3F). Notably, s ome subtitutions on these positions 224 \n(e.g. Ku70 L310R, Ku80 E304A) also affected PAXX recruitment, although at an intermediate 225 \nextent compared to the full defect observed for Pol λ BRCT (Figure 3C-E). Considering that 226 \nPAXX binding to Ku70 is far away from the Ku bridge region, this indicate s that these 227 \nsubstitutions likely compromise the stability of the Ku:DNA interaction in addition to directly 228 \naffecting the Ku :polymerase interaction. Nevertheless, these data establish that Ku70 F303, 229 \nL310 and Ku80 E292, E304 are key positions for Ku interaction with Pol λ. 230 \n 231 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 10 \n 232 \nFigure 3. Impact of mutations in the bridge region of Ku70 and Ku80 on the recruitment of the 233 \nBRCT domain of Pol  at DSBs. A) Position of mutated residues in the bridge domain of Ku70 234 \n(orange) or Ku80 (green) at the interface with Pol  (purple). B) Principle of the laser micro-irradiation 235 \nexperiment. U2OS cells engineered for auxin (IAA) -induced Ku70 knockdown, rescued with WT or 236 \nmutated forms of Ku70 and expressing both the GFP-tagged BRCT domain of Pol  and the mCherry-237 \ntagged PAXX protein, were micro -irradiated and accumulation of each fluorescence was analyzed as 238 \nin Figure 2. C) Representative images before (upper frames) and 20 s after irradiation (lower frames, a 239 \nwhite arrow indicates irradiated area) of nuclei from U2OS expressing either WT Ku70 (left) or the 240 \nL310R mutant (right) D) Quantification of fluorescence accumulation at laser -induced DNA damage 241 \nsites of GFP-Pol-BRCT (upper chart) and mCherry-PAXX (lower chart) in U2OS cells expressing the 242 \nindicated Ku70 constructs. Results are plotted as mean values of at least 20 nuclei ± SEM. E) Principle 243 \n \n \n \n \n \n \n \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 11 \nof the laser micro-irradiation experiment. U2OS cells engineered for doxycycline (doxy)-induced Ku80 244 \nknockdown, rescued with WT or mutants of Ku80 and expressing both the GFP-tagged BRCT domain 245 \nof Pol  and the mCherry -tagged PAXX protein, were micro -irradiated and accumulation of each 246 \nfluorescence was analyzed as in (D). F) Quantification of fluorescence accumulation at laser -induced 247 \nDNA damage sites of GFP-Pol-BRCT (upper chart) and mCherry-PAXX (lower chart) in U2OS cells 248 \nexpressing the indicated Ku80 constructs. Results are plotted as mean values of at least 20 nuclei ± 249 \nSEM. 250 \n 251 \nGap-filling assay in cells to assess impact of Ku70/80:Pol λ interface mutations 252 \nPol λ has a large spectrum of substrates, accommodating breaks with overhangs (<4 to 12 nt), 253 \nmicrohomologies (1-6 nt) and gaps (1-8 nt) 22. Based on these features and to assess the impact 254 \nof mutations at the Ku:Pol λ interface on Pol λ activity during end-joining, we designed a Cas9-255 \ntargeted plasmid , which once transfected in HEK -293T cells can be used as a gap -filling 256 \ndependent DNA repair reporter (Figure 4A). Briefly, the reporter is designed such that the Cas 257 \nfamily member Cpf1 (Cas12a) induces two staggered DSBs in the mCherry cDNA which have 258 \ntwo complementary nucleotides at the very end. Gap -filling at the junction restores mCherry 259 \nexpression, while end-filling enables the expression of a downstream EGFP cDNA which lies 260 \nin a different reading frame. No expression of mCherry or GFP is expected if ends are trimmed 261 \nor not ligated . Expression of BFP from a co -transfected plasmid accounts for transfection 262 \nefficiency. Following Cpf1 -induced DSB, expression of fluorescent proteins is measured by 263 \nflow cytometry. We first established that gap -filling dependent mCherry expression relied on 264 \ncells being transfected by the complete Cpf1/gRNA system (Figure 4B), and on NHEJ activity 265 \nsince it was largely inhibited by a DNA-PK inhibitor catalytic activity (NU7441), or upon Ku 266 \nremoval or genetic ablation of the NHEJ factors DNA-PKcs, LigIV, XLF or PAXX involved 267 \nin break ends tethering and end joining 2 (Figure S7D, E). Moreover, sequencing the junctions 268 \nfrom mCherry positive cells revealed that the expected product from gap-filling repair was 269 \nformed, further validating our reporter system (Figure S7F). Notably, no GFP expression was 270 \ndetected implying that gap -filling is far dominant over end -filling at these staggered DSB 271 \n(Figure 4B). Then, we evaluated the Pol λ-dependency of the gap-filling activity detected. Pol 272 \nλ was found dominant over Pol µ in gap -filling at three-nucleotide gaps on 5′ overhang 273 \nsubstrates 14,23. Indeed, Pol λ knock -out (KO) led to a ~70% reduction of the activity on our 274 \nsubstrate that was fully restored upon re-expression of WT Pol λ, while Pol µ KO alone did not 275 \nimpair gap-filling activity (Figures S7G, H). Notably, Pol µ KO in Pol λ KO cells further 276 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 12 \ndecreased gap-filling activity, suggesting a small compensation by Pol μ in the absence of Pol 277 \nλ. Finally, expression of a catalytically dead Pol λ (D427A-D429A, 24) (Pol λ dead ) further 278 \ndecreased the remaining gap-filling activity in Pol λ KO cells, supporting a dominant negative 279 \neffect of the Pol λ dead construct on backup gap-filling enzymes, including Pol µ. 280 \n 281 \nCharacterization of key positions in Pol λ BRCT:Ku interface for Pol λ-dependent gap-filling 282 \nactivity and survival to IR 283 \nUsing the mainly NHEJ - and Pol λ -dependent gap -filling assay described above, we then 284 \nanalysed the impact of mutations in Pol λ BRCT though transfection of Pol λ KO HEK-293T 285 \ncells complemented with WT or mutant Pol λ. As a control, the same cells were used to evaluate 286 \ndirect end-joining (EJ) activity at blunt-ended breaks using our dedicated Cas9-based reporter 287 \nplasmid assay described previously 19. As shown in Figure 4C, except for R96A, all other 288 \nmutations tested in Pol λ BRCT reduced gap-filling efficiency at staggered DSB. The effect of 289 \nF61G mutation is inconclusive since it lowered the protein expression (Figure S8A). Notably, 290 \nthe extent of defect observed in gap-filling activity of full-length mutant Pol λ correlates with 291 \nthat observed in recruitment of the corresponding mutant GFP -BRCT at laser sites ( Figure 292 \n2E), again with R57 and L60 positions being the most crucial. Since no detectable repair defect 293 \nwas found at blunt-ended DSB, a readout for direct EJ, this indicates that outside its catalytic 294 \nfunction at defined breaks Pol λ is unlikely to fulfill a general function in the overall NHEJ 295 \ncomplex assembly and/or stability. Using the same assays, we then evaluated the impact of 296 \nmutations of Ku70 in the Pol λ:Ku interface by using HEK-293T cells expressing mAID-Ku70 297 \nfusion and transduced with WT or mutated Ku70 forms (Figure S8B). Upon auxin addition, 298 \nwe analysed in parallel the impact of individual or combined mutations in Ku70 on gap-filling 299 \nat staggered DSB and on direct EJ at blunt DSB (Figure 4D). Compared to the slight reduction 300 \nin gap -filling observed with Ku70 mutants at T307 and L310 positions, F303G mutation 301 \nreduced gap -filling by 60% without impairing repair at blunt -ended breaks, indicating that 302 \nKu70 F303 is a crucial position for Ku interaction with Pol λ BRCT but not for Ku interaction 303 \nwith DNA. Then, assessing cell radiosensitivity, we observed that loss of Pol λ marginally 304 \nreduced cell resistance to ionizing radiation ( IR) that was much more decreased upon 305 \nexpression of the Pol λ dead construct ( Figure 4E), in agreement with gap -filling activity 306 \nassessed in parallel (Figure S7H). Notably, the R57E mutation that impaired Pol λ recruitment 307 \nto DSB released the sensitivity associated with expression of the Pol λ dead construct when 308 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 13 \nboth mutations were combined (Figure 4E) and similarly enhanced gap-filling activity up to 309 \nthe level observed in Pol λ KO cells (Figure S8C). Finally, since we observed that the loss of 310 \ncell viability consecutive to Ku removal is reversible within a 32 h time window, we assessed 311 \nthe consequence of impairing Ku :Pol λ interaction on cell survival to IR (Figure S8D and 312 \nFigure 4F). We showed that the Ku70 F303G mutant that preserved direct EJ but impaired 313 \ngap-filling did not fully complement the radiosensitivity observed upon Ku removal. This 314 \nsupports that Ku interaction with gap-filling polymerases promotes cell survival to IR through 315 \npositioning them at the break ends (Figure 4F). 316 \n 317 \n 318 \n \n \n \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 14 \nFigure 4. Effect of Pol  or Ku mutations on gap-filling activity in cells and survival to IR. A) Gap-319 \nfilling reporter substrate. The gap -filling reporter substrate consists of two consecutive frameshifted 320 \n(+1) coding sequences for mCherry (mCh) and EGFP, respectively. The mCherry coding sequence is 321 \ninterrupted by a cassette containing the HSV -TK polyadenylation sequence (TK -pA) flanked by two 322 \ninverted copies of a Cpf1 target sequence (blue characters; the PAM GAAA sequence is shown in 323 \norange). Following Cpf1-mediated double cleavage, the HSV-TK polyadenylation sequence is deleted 324 \nand the resulting 5’ overhang DNA ends can be rejoined in different ways. First, the two overhangs can 325 \npartially anneal to each other by two G:C pairs, leading to single-stranded gaps than can be filled, thus 326 \nrestoring an intact mCherry coding sequence. Second, the two overhangs can undergo end -filling, 327 \nleading to blunt ends whose joining disrupts the mCherry reading frame (+2 frameshift) but enables the 328 \nexpression of the downstream EGFP coding sequence. Finally, the two overhangs can be trimmed off 329 \nbefore end-joining, leading to a -8 frameshift of the mCherry coding sequence and resulting in neither 330 \nred nor green fluorescence. B) Gap-filling assay. Top: the gap -filling reporter assay is performed by 331 \ntransfecting HEK-293T cells with the reporter substrate together with a Cpf1/gRNA-expressing vector 332 \nto cleave the substrate and a BFP -expressing vector to normalize for transfection efficiency. 333 \nFluorescence expression is analyzed 48 h later by flow cytometry. Bottom: representative fluorescence 334 \nmicroscopy images of cells non -transfected (NT) or transfected with the full reporter system (full) or 335 \nomitting the substrate (-substrate) or the gRNA expression vector (-gRNA). PC: phase contrast. C) Gap-336 \nfilling activity (red bars) or direct end-joining activity (orange bars) assessed in parallel in HEK-293T 337 \ncells knocked-out for Pol  and complemented with either an empty vector (EV) or expression vectors 338 \nfor WT or different mutants of Pol . Results are normalized to the WT condition and plotted as mean 339 \nvalues of three to six independent experiments ± SD. P -values from Student’s t-test between the WT 340 \ncondition and the considered mutant, for gap-filling and direct end-joining activities, respectively, are 341 \nas follows: R96A (0.0731; 0.0583), S116G (0.0007; 0.0176), L60A (0.0156; 0.0165), L60R (0.0089; 342 \n0.3312), R57A (<0.0001; 0.0025), R57E (0.0021; 0.0252), F61G (0.0005; 0.0528). * P < 0.05, ** P < 343 \n0.01, *** P < 0.001, **** P < 0.0001, ns: not significant. D) Gap-filling activity (red bars) or direct 344 \nend-joining activity (orange bars) assessed in parallel in HEK-293T cells knocked-down for Ku70 and 345 \nrescued with expression vectors for WT or different mutants of Ku70. Results are normalized to the 346 \nWT condition and plotted as mean values of four experiments ± SD. P -values from Student’s t-test 347 \nbetween the WT condition and the considered mutant, for gap-filling and direct end-joining activities, 348 \nrespectively, are as follows: R301A (0.0541, 0.9381), F303G (<0.0001; 0.0841), T307A (0.0006, 349 \n0.0039), L310G (0.0015; 0.0503), L310R (0.0025; 0.0437). E) Cell survival to X-rays of HEK-293T 350 \ncells knocked-out for POLL and complemented with an empty vector (EV) or expression vectors for 351 \nWT or the indicated mutants of Pol . Y axis is log scale. Results are normalized to the untreated 352 \ncondition and plotted as mean values of four to seven experiments ± SD. P-values were calculated at 2 353 \nGy using unpaired t -test: Pol -dead versus EV (0.0207); Pol -dead versus WT (0.0092). F) Cell 354 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 15 \nsurvival to X -rays of U2OS cells knocked -down for Ku70 (Ku -KD) and complemented or not with 355 \nexpression vectors for WT Ku70 or the F303G mutant. Results are mean values of four experiments 356 \nand plotted as in (E). P-values at 2 Gy: Ku70-F303G versus Ku-KD (0.0195); Ku70-F303G versus Ku-357 \nWT (0.0089). 358 \n 359 \n 360 \nDiscussion 361 \nWe determined the cryo-EM structures of Pol λ in complex with the Ku 70/80 heterodimer 362 \nbound to DNA whilst engaged with the DNA-PK holoenzyme. These structures show a clearly 363 \ndefined interaction between the BRCT domain of Pol λ and the interface between Ku70/80. 364 \nWe assessed the functionality of this interaction by generating specific site directed mutants on 365 \neither Pol λ BRCT or Ku70/80. These mutants were used in two orthogonal assays: live protein 366 \nrecruitment at nuclear laser sites and gap -filling activity in cells transfected with an original 367 \nreporter assay based on Cpf1-generated partially complementary DSB ends. These data define 368 \nthe molecular basis and essentiality of the BRCT domain for recruitment of Pol λ within the 369 \nNHEJ complex. The data also establishes key positions on Ku 70/80 and Pol λ -BRCT that 370 \nmediate their interaction , and for the first time position the interaction region at the external 371 \nface of the Ku70/80 dimer interface. 372 \nFrom our cryo-EM structure of the Ku80 mediated DNA -PK dimer bound to LX4, t he LigIV 373 \ntandem BRCT1 domain can be seen occupying the previously described site on Ku70/80 6,7 374 \nthat is distinct from the Pol λ BRCT interaction site (Figures 1 and 5). The LigIV BRCT1 sits 375 \nin the groove formed by the Ku70/80 dimer interface, whereas Pol λ BRCT is located at an 376 \nadjacent site. From our structural data, it is apparent that the two proteins have distinct sites of 377 \ncontact with Ku70/80, and there is no evidence that Pol λ and LigIV form direct interactions 378 \nwith each other. Thus, the previously reported observation that specific Pol λ mutations prevent 379 \ncomplex formation with Ku70/80 -LigIV-XRCC4 are likely due to disruption of the Pol 380 \nλ:Ku70/80 interaction specifically, as has previously been suggested 16. Interestingly, when we 381 \ncollected cryo-EM data of Ku70/80 alone (without DNA-PKcs) with Pol λ, we did not observe 382 \nany density for the BRCT domain of Pol λ engaged with Ku70/80. Therefore, it is possible that 383 \nthe Pol λ interaction with Ku70/80 is stabilised within the DNA-PK holoenzyme complex. Our 384 \ncryo-EM structures indicate a possible weak interaction between the BRCT domain of Pol λ 385 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 16 \nand DNA-PKcs. Although the resolution is low, we would predict that Pol λ (residues 128-386 \n130) may interact with residues Ser187 and/or Glu188 on DNA-PKcs. 387 \nNotably, we found that recruitment of the N-terminal Pol λ BRCT domain to micro-irradiated 388 \nareas was essentially Ku70/80-dependent in contrast with data obtained with the full-length Pol 389 \nλ. This suggests that Pol λ recruitment to sites of DNA damage may rely on interactions outside 390 \nof its BRCT domain. This may reflect additional roles of Pol λ outside of classical NHEJ such 391 \nas in Base Excision Repair where it is proposed to interact with DNA glycosylases involved in 392 \nthe repair of alkylated or oxidized bases 20. As previously reported, we found that Pol λ 393 \ndeficient cell lines showed modest , if any, sensitivity to IR, whereas cells deficient in both λ 394 \nand µ polymerases were clearly IR sensitive 14,25,26. The expression of a catalytically inactive 395 \nform of Pol λ concurrently inhibited gap -filling activity in cells (this work) and negatively 396 \nimpacted on cell survival to IR as previously reported 24, to extents higher than the sole deletion 397 \nof Pol λ. This suggests that Pol λ dead prevents the rescue of the Pol λ defect by Pol µ, likely 398 \nthrough occupying and occluding a common interaction site on Ku 70/80, and thereby 399 \nsustaining a dominant negative effect. Also, we showed that Ku70 F303G mutant cells exhibit 400 \nsensitivity to IR equivalent to that of a double Pol λ-Pol  KO cells, suggesting again that this 401 \nmutation impairs Ku70/80 interaction with both Pol X proteins. Indeed, AlphaFold prediction 402 \nof the structures formed between Ku70/80 with DNA and the BRCT domains of Pol  and TdT 403 \nindicates the use of similar protein :protein interfaces ( Figure 5B, C). Also, the broad 404 \ncomposition and organization of the Pol λ BRCT domain is conserved amongst Pol X members, 405 \nsupporting a conserved mode of interaction with Ku70/80 (Figure 5D). Together with our 406 \npresent cellular data, this allows us to propose a unified model for the interaction of the three 407 \nBRCT-bearing Pol X with Ku 70/80 on the same site allowing their respective recruitment to 408 \nthe NHEJ complex. 409 \nThe Ku70/80 interaction with Pol X BRCT domain reported here adds to the fascinating list of 410 \nKu70/80 sites that contact NHEJ factors. These include Ku80 sites for A-KBM bearing proteins 411 \n(APLF, WRN, MRI), XLF via its X-KBM 27 and DNA-PKcs 8, Ku70 sites for PAXX via its P-412 \nKBM 2 and Lig IV via its BRCT1 7,8, and combined sites in Ku80/Ku70 for inositol 413 \nhexaphosphate 19 and now for Pol X BRCT s (B-KBM). The structures reported here of the 414 \nKu70/80:Pol λ interaction represents a paradigm for Pol X mode of binding to Ku70/80 and 415 \nreinforces the notion that Ku70/80 acts as a central structural hub in the NHEJ mechanism 28. 416 \nLike other Ku70/80 partners whose KBM motifs are at the N or C-terminus of the protein, Pol 417 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 17 \nX members are anchored by an N-terminal BRCT domain separated by a flexible region from 418 \nthe catalytic portion of the protein , allowing free movement for the correct positioning at the 419 \nsite of the DSB. 420 \nThe structures of the LRC presented here indicate that the Pol X:Ku70/80 interaction occurs 421 \nearly during the NHEJ process, at a stage where DNA -PKcs is still present and at which the 422 \nDNA ends are likely not yet accessible to processing enzymes. Moreover, since our cryo -EM 423 \nstructures were obtained using a blunt -ended DNA substrate, this suggests that it is not the 424 \nnature of the DNA ends that dictates Pol X engagement into the NHEJ process but rather its 425 \nintrinsic affinity for the initial DNA -PK complex. These structures also illustrate that the 426 \nevolution of distinct and independent binding sites on the Ku70/80 heterodimer enables the 427 \nconcurrent anchoring of NHEJ proteins (PAXX/XLF, DNA -PKcs, Pol X, XRCC4/LigIV) 428 \ndedicated to specific activities involved in the repair reaction (synapsis, kinase, polymerase, 429 \nligase, respectively ) (Figure 5E). Moreover, the dimeric nature of the LRC allows for a 430 \nscenario where two different members of Pol X family could be engaged within the same 431 \ndimeric complex simultaneously. Although our DSB repair assay in cells could theoretically 432 \nreport both gap-filling and end-filling activities, we found that gap-filling is far dominant. This 433 \nsuggests that, when possible, end -annealing on minimal homology is preferred, most likely 434 \nbecause it contributes to the stability of the synaptic complex and ultimately to the fidelity of 435 \nDNA repair . The two -nucleotide overhang substrate used here supports a tight coupling 436 \nbetween end annealing and gap filling, as demonstrated elegantly for NHEJ mediated repair in 437 \nXenopus egg extracts 23. Within the NHEJ LRC, this coupling is ensured by the concurrent 438 \nbinding of ends , and resynthesis factors by Ku70/80 on both sides of the DSB. Also, the 439 \ncoexistence of LigIV and processing enzymes like Pol X in the synaptic complex at break ends 440 \nensures that ligation can proceed as soon a DNA ends are ligatable, limiting unnecessary DNA 441 \nsequence alteration 23. This illustrates that the unique capability of Ku 70/80 for simultaneous 442 \nmultivalent interaction is crucial for the high adaptability of the NHEJ process 29. 443 \n 444 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 18 \n 445 \nFigure 5: Comparison of the binding of BRCT domains from NHEJ polymerases X ( ,  and 446 \nTdT) to Ku70/80 and scheme of Ku as a structural hub. A) Experimentally determined structure 447 \nfrom this work showing Pol  binding to Ku70/80, DNA and PAXX. B) AlphaFold 3 prediction of Pol 448 \n binding to Ku70/80 and DNA. C) AlphaFold 3 prediction of Pol TdT binding to Ku70/80 and DNA. 449 \nKu70 is in orange, Ku80 in green, DNA in yellow, PAXX P-KBM in cyan and Polymerase BRCTs in 450 \npurple. D) Sequence alignment of the BRCT domain of Pol X family members generated by ESPript 30. 451 \nStructural elements from the Pol  cryo-EM structures are shown above the alignment. Residues 452 \nmutated in this study are indicated with *. E) Ku70/80 (orange/green) as a structural hub showing the 453 \nbinding of Pol  (purple), Ligase IV (red), PAXX (cyan), XLF (pink) and APLF (brown). 454 \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 19 \nAcknowledgements: We thank Dr Christos Savva, Dr Emma Hesketh, Dr Claudia Lancey and 455 \nDr TJ Ragan from the Midlands Regional Cryo -EM facility for help with grid preparation, 456 \nscreening, data collections and processing support. P.F., N.B., J.C., S.B. V.R, J.B.C and P.C. 457 \nwere supported by the French National Research Agency ( ANR-20-CE11-0026). This work 458 \nwas supported by the Fondation ARC (J.C.). P.C. is a scientist from INSERM. We 459 \nacknowledge the imaging facility TRI, member of the national infrastructure France -460 \nBioImaging infrastructure supported by the French National Research Agency (ANR -10-461 \nINBS-04). JB.C and V.R thank the I2BC Protex platform supported by French Infrastructure 462 \nfor Integrated Structural Biology (FRISBI) ANR-10-INBS-0005. J.C and V.R were supported 463 \nby ANR-21-CE12-0019-01, ANR-22-CE12-0037 and ANR 23-CE11-0033. 464 \nFunding: We thank the Lister Institute of Preventative Medicine Prize for support of this 465 \nresearch. We would also like to thank the Medical Research Council for the standard research 466 \ngrant (MR/X00029X/1). 467 \nAuthor contributions: 468 \nA.K.C. directed the study. A.K.C. , P.C. and P.F. led the experimental design. H.A., S.Z., 469 \nA.K.C., S.B., P.F. and P.C. prepared the manuscript. H.A. and S.Z. collected the cryo-EM data 470 \nand modelled and analysed the structures. C.H . helped with structural analysis and edited 471 \nmanuscript. G.M. Purified proteins and collected initial cryo-EM data. P.F. designed and built 472 \nthe cellular tools. P.F. and N.B. designed, validated and carried out the in vivo repair 473 \nexperiments. P.F. and J.C. performed the multiphoton laser micro -irradiation experiments. 474 \nA.K.C., S.B. and P.C. acquired funding. S.W.H and D.Y. C advised with cryo -EM data 475 \ncollection set -up and processing . S.W.H helped with structural analysis and manuscript 476 \npreparation. V.R and J.B.C provided insect cells pellets for human NHEJ factors. 477 \nCompeting interests: 478 \nThe authors declare no competing interests. 479 \nData and materials availability: All structural data presented are publicly available. Cryo -480 \nEM structures and maps are deposited at the PDB and EMDB with accession codes as follows: 481 \n 482 \n 483 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 20 \nMaterials and Methods 484 \nPurification of DNA-PKcs and Ku70/80 485 \nDNA-PKcs and full -length His -tagged Ku70/80 were expressed and purified as previously 486 \ndescribed 6. 487 \nExpression and purification of full-length XLF and LX4 488 \nA construct containing full -length 10xHis-tagged XLF and LX4 (Lig IV and XRCC4) were 489 \nexpressed in insect cells and purified as previously reported 2. 490 \nDNA annealing. Biotinylated Y-shaped 42-55 bp dsDNA were synthesised and annealed as 491 \ndescribed previously 6. DNA sequences used for annealing can be found below. 492 \nY-shaped DNA Forward 493 \nBiotin-494 \nCGCGCCCAGCTTTCCCAGCTAATAAACTAAAAACTATTATTATGGCCGCACGCGT 495 \nY-shaped DNA Reverse 496 \nACGCGTGCGGCCATAATAATAGTTTTTAGTTTATTGGGCGCG 497 \nCryo-EM sample preparation of DNA -PK, LX4, PAXX and Polymerase  structure 498 \nProteins were concentrated using a centrifugal filter (Amicon) with a 30 kDa cut-off and buffer 499 \nexchanged into 20 mM HEPES, pH 7.6, 200 mM NaCl, 0.5 mM EDTA, 2 mM MgCl 2, 5 mM 500 \nDTT. Purified Ku70/80 full-length was then first mixed with Y-shaped 42-55 bp DNA before 501 \nbeing mixed with purified DNA -PKcs, LX4, PAXX and Pol  in respectively a 1:1:2:2:2:6 502 \n(DNA:DNA-PKcs:Ku:LX4:PAXX:Pol) ratio. 503 \nCryo-EM grid preparation 504 \nAliquots of 3 μl of ~2.5 mg/ml of the NHEJ complex (based on DNA -PKcs concentration) 505 \nwere mixed with 8 mM CHAPSO to eliminate particle orientation bias (final concentration ; 506 \nSigma) before being applied to Holey Carbon grids (Quantifoil Cu R1.2/1.3, 300 mesh), glow 507 \ndischarged for 60 s at current of 25 mA in PELCO Easiglow (Ted Pella, Inc .). The grids were 508 \nthen blotted with filter paper once to remove any excess sample, and plunge -frozen in liquid 509 \nethane using a FEI Vitrobot Mark IV (Thermo Fisher Scientific) at 4 oC and 95 % humidity. 510 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 21 \nCryo-EM data acquisition 511 \n The data was collected on a Titan Krios equipped with a Gatan K3 direct electron counting 512 \ndetector at the University of Leicester. All data collection parameters are given in Table S1. 513 \nCryo-EM Image processing 514 \nThe classification process for the two datasets is summarised schematically in Figure S2. The 515 \nfinal reconstructions obtained had overall resolutions (Table S1), which were calculated by 516 \nFourier shell correlation at 0.143 cut-off. 517 \nCryo-EM structure refinement and model building 518 \nThe model of the DNA -PK monomer (PDB:7NFE) was used as an initial template and rigid -519 \nbody fitted into the cryo-EM density in UCSF chimera 31 and manually adjusted and rebuilt in 520 \nCoot 32. LX4 was either removed or manually adjusted depending on whether density was 521 \npresent and PAXX was docked within the central density. The BRCT domain of Polymerase  522 \nwas then manually docked into the density ( PDB: 2JW5 ) in chimera. Models and their 523 \ncorresponding density maps were run through Namdinator 33 before being refined using Phenix 524 \nreal-space refinement 34. 525 \nCell lines, cell culture and cell engineering 526 \nU2OS cells (human osteosarcoma cell line from ECACC, Salisbury, UK) and HEK -293T 527 \nhuman embryonic cells, were grown in DMEM (Eurobio, France) supplemented with 10% fetal 528 \ncalf serum (Eurobio, France), 125 U/ml penicillin, and 125 μg/ml streptomycin. Cells were 529 \nmaintained at 37°C in a 5% CO2 humidified incubator. 530 \nHEK-293T cells knocked-out for POLL (DNA Polymerase ), POLM (DNA Polymerase ), 531 \nPRKDC (DNA -PKcs, Addgene Plasmid#220493 35, LigIV, XLF and PAXX genes were 532 \nobtained following cell transfection with the pCAG-eCas9-GFP-U6 vector expressing the 533 \ncorresponding guide RNA (see below) using jetPEI (Polyplus) as a transfection reagent. 534 \nFollowing cell sorting, individual clones were isolated and checked by western blot. 535 \nThe generation of U2OS cells expressing an inducible shRNA against Ku80 and the generation 536 \nof U2OS and HEK-293T cells expressing a mini-auxin-inducible degron-tagged Ku70 protein 537 \n(mAID-Ku70) in place of the endogenous Ku70 protein have been previously described 19,36. 538 \nProduction of lentiviral particles in HEK-293T cells and transduction of U2OS and HEK-293T 539 \ncells were performed as previously described 37. 540 \n 541 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 22 \nWestern-blot and antibodies 542 \nCell pellets were washed with phosphate-buffered saline (PBS) and resuspended in lysis buffer 543 \n(50 mM Hepes-KOH, pH 7.5, 450 mM NaCl, 1 mM EDTA, 1% Triton X -100) supplemented 544 \nwith Halt protease inhibitor cocktail (ThermoFisher Scientific). Cells were lysed by four 545 \nfreeze/thaw cycles in liquid nitrogen and 37°C water bath. Lysates were cleared by 546 \ncentrifugation and protein concentrations were determined using the Bradford assay (Bio-Rad, 547 \nHercules, CA). Equal amounts of proteins were mixed with concentrated loading sample buffer 548 \nto 1X final concentration (50 mM Tris.HCl pH 6.8, 10 % glycerol, 1 % SDS, 300 mM 2 -549 \nmercaptoethanol, 0.01 % bromophenol blue), heat -denatured, separated by SDS -PAGE on 550 \nMiniprotean TGX stain -free 4-15 % gradient gels (Bio -Rad, Hercules, CA) and blotted onto 551 \nProtran 0 .45 µm nitrocellulose membranes (GE Healthcare) . Membranes were blocked for 552 \n60 min with 5% non -fat dry milk in PBS, 0.1% Tween -20 (Sigma-Aldrich) (PBS-T buffer), 553 \nincubated as necessary with primary antibody diluted in PBS -T containing 1% bovine serum 554 \nalbumin (immunoglobulin- and lipid-free fraction V; Sigma-Aldrich) and washed 3 times with 555 \nPBS-T. Membranes were incubated for 1 h with HRP -conjugated secondary antibodies 556 \n(Jackson Immunoresearch Laboratories) in PBS -T and washed five times with PBS -T. 557 \nImmuno-blots were visualized by enhanced chemiluminescence (Western Lightning Plus -558 \nECL; Perkin Elmer) and autoradiography. Primary antibodies used: mouse monoclonal 559 \nantibodies anti-DNA-PKcs (clone 18.2; Thermo Fisher Scientific), anti-Ku80 (clone 111), anti-560 \nKu70 (clone N3H10), anti -Pol lambda (clone E11, Santa Cruz), anti -beta-Actin (clone C4, 561 \nSanta Cruz); rabbit monoclonal antibodies an ti-LigIV (A11432, Abclonal ), anti -Pol mu 562 \n(EPR10470(B), Abcam); rabbit polyclonal antibodies anti -XLF (A199957, Abclonal), anti -563 \nPAXX (NBP1-94172, Novus). 564 \n 565 \nIonizing irradiation and cell survival analysis 566 \n Two to six thousand U2OS or HEK -393T cells per well were seeded in duplicate in six -well 567 \nplates. Cells were exposed 24 h later to various doses of X-ray using a Faxitron RX-650 device 568 \n(130 kV, 5 mA, dose rate 0.3 Gy per min). Six to seven days later, cells were fixed with 7% 569 \ntrichloroacetic acid for 1 h at 4°C. Fixed cells were extensively washed with water and plates 570 \nwere air dried before staining for 15 min with crystal violet (0.1% aqueous solution). Stained 571 \ncells were further extensively washed with water and plates were air dried. Staining was 572 \ndissolved with 10% acetic acid solution and absorption was measured at 570 nm (Ultrospec -573 \n3000 spectrophotometer, Pharmacia Biotech). Results were plotted as mean values of >3 574 \nindependent experiments ± s.d. using Microsoft Excel software. 575 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 23 \n 576 \nMultiphoton laser micro-irradiation 577 \nLive cell microscopy and multiphoton laser micro -irradiation were conducted as previously 578 \ndescribed 27. 579 \n 580 \nIn vivo DNA end-joining assays 581 \nTo assess gap -filling activity, HEK -293T cells were seeded to 20 -40% confluence in 6 -well 582 \nplates and transfected 24 h later with a mix of Cpf1 -targeted gap-filling reporter substrate, 583 \nCpf1/gRNA expressing vector and mTagBFP2 expressing plasmid as an internal control. Cells 584 \nwere trypsinized 2 days post -transfection, washed with PBS and analyzed by flow cytometry 585 \non a Fortessa X -20 cell analyzer (BD Biosciences). The integrated red fluorescence signal 586 \naccounting for gap-filling-mediated repair events (% positive cells x mean fluorescence) was 587 \nnormalized to that of transfection efficiency (BFP). For the repair junction analysis, a variant 588 \nof the substrate vector was generated in which a sequence encoding HygroR-T2A was inserted 589 \nin frame, upstream of the disrupted mCherry sequence. This plasmid was stably transfected 590 \ninto HEK -293T cells and, after selection with hygromycin, positive clones were isolated. 591 \nFollowing further transient transfection with the Cpf1/gRNA expressing vector, red fluorescent 592 \nand non-fluorescent clones were isolated. Sequence junctions were analyzed by genomic DNA 593 \nextraction (SV Genomic DNA Purification System, Promega), PCR amplification with primers 594 \nmCh-Xba-F and mCh-Xho-R and sequencing (Eurofins Genomics; Ebersberg, Germany). 595 \nDirect end -joining activity was assessed as described previously with a dedicated reporter 596 \nsubstrate in which blunt -ended DSBs were generated with appropriate Cas9/gRNA co -597 \nexpression 19. 598 \n 599 \nPlasmids and DNA manipulations 600 \nCas9 and gRNA expressing vectors for gene knockout were generated by inserting the pre -601 \nannealed gRNA-F and gRNA-R oligonucleotides of the corresponding targeted gene into the 602 \nBbsI restriction sites of pCAG-eCas9-GFP-U6-gRNA plasmid (a gift from Jizhong Zou, 603 \nAddgene plasmid # 79145 ; http://n2t.net/addgene:79145 ; RRID:Addgene_79145). See below 604 \nthe list of oligonucleotides. 605 \nThe previously described pLV3 lentiviral vector 27 was modified as follows to allow insertion 606 \nof Pol  cDNA downstream a puromycin resistance gene and a sequence encoding the T2A 607 \nribosomal skipping peptide: first, a T2A cassette (pre -annealed oligonucleotides kpn2 -T2A-608 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 24 \nMlu-F and kpn2 -T2A-Mlu-R) was inserted into the Kpn2I and MluI restriction sites of the 609 \npLV3 plasmid; second, a PCR -amplified fragment with a Puro -resistance cDNA sequence 610 \n(PCR reaction with primers HF -Puro-F and HF -Puro-R on a synthetic DNA molecule as a 611 \ntemplate) was added by Hot -Fusion 38 at the Kpn2I site, resulting in the pLV3 -Puro-T2A 612 \nplasmid. A PCR-amplified human Pol  cDNA fragment (primers PolL-Mlu-F and PolL-Bcu-613 \nR) was then inserted between the MluI and BcuI restriction sites of pLV3-Puro-T2A. 614 \nExpression vectors for mutant forms of Pol  were obtained in a similar manner following an 615 \nadditional step of overlap extension PCR mutagenesis with the corresponding PolL-mut-F and 616 \nPolL-mut-R oligonucleotides as mutated inner primers (see below the list of primers). 617 \nThe expression vector for GFP-tagged Pol  constructs (full-length protein or BRCT domain) 618 \nwas obtained by replacing the FLAG-Ku70 cDNA from the previously described pLV3-GFP-619 \nFLAG-Ku70 vector 2 by a linker cassette (pre-annealed Kpn2-AX-Mlu-F and Kpn2-AX-Mlu-620 \nR oligonucleotides) between the Kpn2I and MluI sites. The resulting pLV3-GFP plasmid was 621 \nthen used to insert between the MluI and BcuI sites the PCR -amplified human Pol  cDNA 622 \nfragment described above. Expression vectors for GFP -tagged WT and mutant Pol  BRCT 623 \ndomain (aminoacids 1-136) were obtained in a similar manner following a PCR amplification 624 \nof the corresponding cDNAs with PolL-Mlu-F and PolL-BRCT-Bcu-R primers and full-length 625 \nPol  expression vectors as templates. 626 \nThe lentiviral vector allowing expression of mCherry-tagged human PAXX was obtained, first, 627 \nby inserting between Kpn2I and MluI restriction sites of pLV3 the coding sequence of 628 \nmCherry, following PCR amplification from the pmCherry -NLS plasmid (a gift from Martin 629 \nOffterdinger; Addgene plasmid # 39319 ; http://n2t.net/addgene:39319 ; 630 \nRRID:Addgene_39319) with primers mCh -Kpn2-F and mCh -Mlu-R, second, by further 631 \ninserting between MluI and BcuI restriction sites the coding sequence of PAXX, following 632 \nPCR amplification from the previously described pLV3 -GFP-PAXX plasmid 2 with primers 633 \nPAXX-Mlu-F and PAXX-Bcu-R. 634 \nKu70 lentiviral expression vector was generated by PCR amplification of human Ku70 cDNA 635 \nwith Ku70 -Kpn2-F and Ku70 -Bcu-R primers and subsequent insertion between Kpn2I and 636 \nBcuI restrictions sites of the pLV3 vector. Expression vectors for mutant forms of Ku70 were 637 \nobtained in a similar manner following an additional step of overlap extension PCR 638 \nmutagenesis with the corresponding Ku70-mut-F and Ku70-mut-R oligonucleotides as mutated 639 \ninner primers (see below the list of primers). 640 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 25 \nKu80 lentiviral expression vector was already described 19. Expression vectors for mutant 641 \nforms of Ku80 were generated following an additional step of overlap extension PCR 642 \nmutagenesis with the corresponding Ku80-mut-F and Ku80-mut-R oligonucleotides as mutated 643 \ninner primers (see below the list of primers). 644 \nThe gap -filling reporter substrate was assembled into the pEGFP -N1 vector (Clontech) 645 \nfollowing sequential insertion of various PCR products, oligonucleotide linkers and synthetic 646 \nDNA fragments (see Figure 4A for a detailed description). The Cpf1 and guide RNA co -647 \nexpression plasmid used to cleave the reporter substrate was generated by inserting the pre -648 \nannealed gRNA-GF-F and gRNA -GF-R oligonucleotides into the Esp3I restriction sites of 649 \npTE4398 (a gift from Ervin Welker ; Addgene plasmid # 74042 ; http://n2t.net/addgene:74042 650 \n; RRID:Addgene_74042). 651 \nThe pNLS -mTagBFP2 plasmid used as an internal control of transfection efficiency was 652 \nobtained by PCR amplification of the 2xNLS -mTagBFP2 coding sequence with mTagBFP -653 \nAcc65-F and mTagBFP -Mlu-R primers on the pHAGE -TO-nls-st1dCas9-3nls-3XTagBFP2 654 \nplasmid template (a gift from Thoru Pederson ; Addgene plasmid # 64512; 655 \nhttp://n2t.net/addgene:64512 ; RRID:Addgene_64512). The resulting PCR fragment was 656 \ninserted into the pEGFP -N1 (Clontech) vector backbone after modification of the multiple 657 \ncloning site and removal of the GFP coding sequence. 658 \nAll oligonucleotides were purchased from Eurofins Genomics (Ebersberg, Germany). 659 \nRestriction and modifying enzymes (Phusion and T4 DNA Ligase) were from ThermoFisher 660 \nScientific (Illkirch, France). All constructs were checked by sequencing (Eurofins Genomics). 661 \n 662 \nOligonucleotides (DNA linkers and PCR primers) 663 \ngRNA-GF-F agatCAACGTACGGTCTGATATGCAA \ngRNA-GF-R aaaaTTGCATATCAGACCGTACGTTG \ngRNA-LigIV-F caccGTTCAGCACTTGAGCAAAAG \ngRNA-LigIV-R aaacCTTTTGCTCAAGTGCTGAAC \ngRNA-PAXX-F caccgTGACCGACGCCGCGGAGCTT \ngRNA-PAXX-R aaacAAGCTCCGCGGCGTCGGTCAc \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 26 \ngRNA-PRKDC-F caccGGTACCCACCCAGCACCGCG \ngRNA-PRKDC-R aaacCGCGGTGCTGGGTGGGTACC \ngRNA-POLL-F caccgTTCACCAGCTGAGCACCCGG \ngRNA-POLL-R aaacCCGGGTGCTCAGCTGGTGAAc \ngRNA-POLM-F caccGATGGAAGAGACCTCAGCAG \ngRNA-POLM-R aaacCTGCTGAGGTCTCTTCCATC \ngRNA-XLF-F caccGGAGATTATCCAAATGACAG \ngRNA-XLF-R aaacCTGTCATTTGGATAATCTCC \nHF-Puro-F GCCTCGAGGTTTAAACTACGGgatcTCCGCcATGACaGAGTACAAGCCaACaGTG \nHF-Puro-R CCGCATGTTAGCAGACTTCCTCTGCCCTCGGCACCtGGCTTtCtGGTCATGCACC \nKpn2-AX-Mlu-F ccggTCAGGATCTGGTAGCGGTTCCGGATCTCCTAGGTCACCCGGGTCTA \nKpn2-AX-Mlu-R cgcgTAGACCCGGGTGACCTAGGAGATCCGGAACCGCTACCAGATCCTGA \nkpn2-T2A-Mlu-F \nCCGGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTG\nGACCCGGGtcactcA \nkpn2-T2A-Mlu-R \nCGCGTgagtgaCCCGGGTCCAGGATTCTCCTCGACGTCACCGCATGTTAGCAGAC\nTTCCTCTGCCCT \nKu70-Kpn2-F ctctcgTCCGGAGCCACCATGTCAGGGTGGGAGTCATATTACAAAACC \nKu70-Bcu-R ctcgtcACTAGTTCAGTCCTGGAAGTGCTTGGTGAGGGCTTC \nKu70-R301A-F GAAAACCAAGACCgcGACCTTTAATACAAGTACAGGCGGTTTGCT \nKu70-R301A-R CTTGTATTAAAGGTCgcGGTCTTGGTTTTCACTGGTTCATTTGT \nKu70-F303G-F CCAAGACCCGGACCggTAATACAAGTACAGGCGGTTTGCTTCTGC \nKu70-F303G-R GTACTTGTATTAccGGTCCGGGTCTTGGTTTTCACTGGTTCATTT \nKu70-T307A-F CGGACCTTTAATACAAGTgCAGGCGGTTTGCTTCTGCCTAGCGATACC \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 27 \nKu70-T307A-R GAAGCAAACCGCCTGcACTTGTATTAAAGGTCCGGGTCTTGGTTTTCAC \nKu70-L310G-F CCTTTAATACAAGTACAGGCGGTggGCTTCTGCCTAGCGATACCAAGAGGTC \nKu70-L310G-R CTAGGCAGAAGCccACCGCCTGTACTTGTATTAAAGGTCCGGGTCTTGG \nKu70-L310R-F CCTTTAATACAAGTACAGGCGGTcgGCTTCTGCCTAGCGATACCAAGAGGTC \nKu70-L310R-R CTAGGCAGAAGCcgACCGCCTGTACTTGTATTAAAGGTCCGGGTCTTGG \nKu80-R292A-F GAAGATATACAAAAAGcAACAGTTTATTGCTTAAATGATGATGATGAAAC \nKu80-R292A-R GCAATAAACTGTTgCTTTTTGTATATCTTCTTTTTTTAGGGTTTTTGC \nKu80-D301A-F GCTTAAATGATGATGcTGAAACTGAAGTTTTAAAAGAGGATATTATTCAAG \nKu80-D301A-R TAAAACTTCAGTTTCAgCATCATCATTTAAGCAATAAACTGTTTCTTTTTG \nKu80-E304A-F GATGATGATGAAACTGcAGTTTTAAAAGAGGATATTATTCAAGGGTTCCGC \nKu80-E304A-R CCTCTTTTAAAACTgCAGTTTCATCATCATCATTTAAGCAATAAACTGTTTC \nKu80-E304R-F GATGATGATGAAACTcgAGTTTTAAAAGAGGATATTATTCAAGGGTTCCGC \nKu80-E304R-R CCTCTTTTAAAACTcgAGTTTCATCATCATCATTTAAGCAATAAACTGTTTC \nmCh-Kpn2-F ctcgtcTCCGGAGCCACCATGGTGAGCAAGGGCGAGGAGG \nmCh-Mlu-R ctcctcACGCGTCTTGTACAGCTCGTCCATGCCGCC \nmCh-Xba-F cctctgTCTAGAgTCCAAGGGCGAAGAAGATAATATGGC \nmCh-Xho-R cctgtgCTCGAGcaTCACTTGTAAAGTTCGTCCATTCCACC \nmTagBFP-Acc65-F ctgtctGGTACCTGGAAGTGGAAGCCCAAAGAAAAAGC \nmTagBFP-Mlu-R ctgtcgACGCGTTTCAATTAAGCTTGTGCCCCAGTTTGCTAGGG \nPAXX-Mlu-F ctcctgACGCGTTCTGGTAGCGGTTCAGGACTCAGATCCATGGACCCGCTGTC \nPAXX-Bcu-R cctctcACTAGTTTAGGTCTCATCGAAGTCCACGCCACCAG \nPolL-Mlu-F ctcctcACGCGTATGGAcCCCAGGGGTATCTTGAAGGCATTTC \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 28 \n 664 \n 665 \n 666 \n 667 \n 668 \nPolL-Bcu-R cctctcACTAGTTCACCAGTCCCGCTCAGCAGGTTC \nPolL-BRCT-Bcu-R cctgacACTAGTtcaACTGGGGATGAAGATGCTGAATCCAGCTACATC \nPolL-dead-F GGCGACCTGTGGTGcTGTCGcCGTGCTCATCACTCACCCAGATGGCC \nPolL-dead-R GAGTGATGAGCACGgCGACAgCACCACAGGTCGCCTTTCCCCGTC \nPolL-R57A-F CATTGGACGAGCCgcGGCAGAACTCTTTGAGAAGCAGATTGTTC \nPolL-R57A-R CAAAGAGTTCTGCCgcGGCTCGTCCAATGCCAGTGCGCACAAC \nPolL-R57E-F CATTGGACGAGCCgaGGCAGAACTCTTTGAGAAGCAGATTGTTC \nPolL-R57E-R CAAAGAGTTCTGCCtcGGCTCGTCCAATGCCAGTGCGCACAAC \nPolL-L60A-F CCGGGCAGAAgcCTTTGAGAAGCAGATTGTTCAGCATGGC \nPolL-L60A-R CTGCTTCTCAAAGgcTTCTGCCCGGGCTCGTCCAATGCCAGTGC \nPolL-L60R-F CCCGGGCAGAACgCTTTGAGAAGCAGATTGTTCAGCATGGC \nPolL-L60R-R CTGCTTCTCAAAGcGTTCTGCCCGGGCTCGTCCAATGCCAGTGC \nPolL-F61G-F CGGGCAGAACTCggTGAGAAGCAGATTGTTCAGCATGGC \nPolL-F61G-R CAATCTGCTTCTCAccGAGTTCTGCCCGGGCTCGTCCAATGC \nPolL-R96A-F GAGCGAGCCCTCgcCCTTCTCAGACTACCCCAGCTGCC \nPolL-R96A-R GTCTGAGAAGGgcGAGGGCTCGCTCATAGTCCATGCC \nPolL-S116G-F GAAGTCAGCCTGGCTGgGCTTGTGCCTTCAGGAGAGGAGGCTGG \nPolL-S116G-R CCTGAAGGCACAAGCcCAGCCAGGCTGACTTCACCAGC \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 29 \nSupplementary Information 669 \nTable 1: Cryo-EM data parameters and statistics 670 \n 671 \n 672 \n 673 \n 674 \n 675 \n 676 \n 677 \n 678 \n 679 \n \n PK-LigIV-PolL \n \nPDB: 9GD7 \nEMD: 51249 \nPK-PolL \n \nPDB: 9G9L \nEMD: 51156 \nData collection and processing \nDetector Gatan K3 Gatan K3 \nMagnification 130k 130k \nEnergy filter slit width (eV) 20 20 \nVoltage (kV) 300 300 \nFlux on detector (e/pix/sec) 21.24 21.24 \nElectron exposure on sample (e–/Å2) 51.96 51.96 \nTarget defocus range (μm) 0.8-2.2 0.8-2.2 \nCalibrated pixel size (Å) 0.652 (bin 1x to 1.304) 0.652 (bin 1x to 1.304) \nSymmetry imposed C1 C1 \nExtraction box size (pixels) 320 320 \nInitial particle images (no.) 603711 603711 \nFinal particle images (no.) 14964 12656 \nRefinement \nMap resolution at FSC=0.143 (Å)* 4.25 4.63 \nModel composition \n Non-hydrogen atoms 41717 39800 \n Protein residues 5158 4944 \n Nucleotides 50 47 \nB factor (Å2) \n Protein 387.65 515.18 \n DNA 354.09 507.46 \nR.m.s deviations \n Bond lengths (Å) 0.004 0.003 \n Bond angles (°) 0.827 0.803 \nValidation \n Molprobity score 2.24 2.34 \n Clashscore 18.8 23.7 \n Poor rotamers (%) 0 0.07 \nRamachandran plot \n Favored (%) 92.53 92.44 \n Allowed (%) 7.25 7.34 \n Disallowed (%) 0.22 0.22 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 30 \n 680 \n 681 \n 682 \n 683 \n 684 \n 685 \n 686 \nFigure S1: Domain organization of Pol X family DNA polymerases. The Pol  constructs used in 687 \nthis study correspond to the full -length protein (FL) and the amino -terminal region (residues 1 -136) 688 \ncontaining the nuclear localization sequence (NLS) and the BRCT domain (BRCT). 689 \n 690 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 31 \n 691 \nFigure S2: Single-particle cryo-EM image processing workflow for DNA-PK +PAXX + Pol  with 692 \nand without LX4. Schematic showing particle picking using WARP and processing including 2D 693 \nclassification and ab initio reconstruction using CryoSPARC. The two main classes generated with the 694 \ncorresponding number of particles is shown and the two maps following non-uniform refinement with 695 \nresolutions for an FSC of 0.143 are given. Additional focused and composite maps are also shown. 696 \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 32 \n 697 \nFigure S3: Cryo-EM data of DNA-PK with BRCT domain of Pol . a) Example of 2d classes. b) 698 \nLocal resolution map of DNA -PK dimer with BRCT domain of Pol  consensus cryo-EM map. c) 699 \nAngular distribution calculated in cryoSPARC for particle projections shown as a heat map of the 700 \nconsensus map. d) FSC resolution curves and viewing distribution plot of the consensus map. e) DNA-701 \nPK dimer with BRCT domain of Pol  consensus cryo-EM map with masking area. f) Local resolution 702 \nmap of DNA -PK dimer with BRCT domain of Pol  locally refined map. g) Angular distribution 703 \ncalculated in cryoSPARC for particle projections shown as a heat map of the locally refined map. h) 704 \nFSC resolution curves and viewing distribution plot of the locally refined map. The colours 705 \ncorresponding to each resolution are displayed on the specific key chart below the maps. 706 \n 707 \n 708 \n 709 \n 710 \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 33 \n 711 \nFigure S4: Cryo-EM data of DNA -PK with BRCT domain of Pol  and LX4. a) Example of 2d 712 \nclasses. b) Angular distribution calculated in cryoSPARC for particle projections shown as a heat map 713 \nof the consensus map. c) Local resolution map of DNA -PK dimer with BRCT domain of Pol  and 714 \nLigase IV consensus cryo -EM map. d) FSC resolution curves and viewing distribution plot of the 715 \nconsensus map. e) and f) Consensus map with masking area 1 and 2, respectively. g) and h) Local 716 \nresolution of the two locally refined maps. i) and f) FSC resolution curves and viewing distribution plot 717 \nof local maps 1 and 2. The colours corresponding to each resolution are displayed on the specific key 718 \nchart below the maps. 719 \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 34 \n 720 \nFigure S5: Comparison of cryo -EM maps. A) DNA-PK + Pol  (this work) in blue. B) DNA-PK 721 \n(PDB: 6ZHA), C) Comparison overlay of DNA-PK + Pol  (blue) and DNA-PK (grey). 722 \n 723 \n 724 \nFigure S6: (A) Western blot on whole cell protein extracts from U2OS cells either unmodified (lane 1) 725 \nor constitutively expressing an shRNA against endogenous Ku70 and rescued with expression of 726 \nmAID-tagged Ku70 (lanes 2-4), treated or not with auxin (IAA) for 16 h. Asterisk indicates the position 727 \nof mAID -Ku70 signal (MW: 77.4 kDa) below that of Ku80. (B) Fluorescence micrographs of 728 \nU2OS/mAID-Ku70 cells (see (A)) expressing full -length GFP-tagged Pol l, in the presence (+Ku) or 729 \nthe absence (-Ku). (C) Fluorescence micrographs of U2OS cells expressing WT or mutated GFP-tagged 730 \nPol l BRCT domain. 731 \n \n \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 35 \n 732 \nFigure S7: (A) Western blot on whole cell protein extracts from U2OS/mAID-Ku70 cells (see 733 \nSuppl. Figure S6A) depleted of endogenous Ku70 and complemented with ectopic expression 734 \nof either WT or mutated forms of Ku70, as indicated. (B) Western blot on whole cell protein 735 \nextracts from U2OS cells either unmodified (lane 1) or expressing a doxycycline (doxy) -736 \ninduced shRNA against endogenous Ku80 (lanes 2 -4) and rescued with expression of Ku80 737 \n(lane4). (C) Western blot on whole cell protein extracts from U2OS/Tet-shKu80 cells (see (B)) 738 \ndepleted of endogenous Ku80 and complemented with ectopic expression of either WT or 739 \nmutated forms of Ku80, as indicated (D) Gap-filling activity assessed in HEK -293T/mAID-740 \nKu70 cells knocked -down for Ku70 when indicated (+IAA), in the presence or not of 3 µM 741 \nDNA-PK inhibitor (+NU7441 or +NU). Results are normalized to the control condition (full) 742 \nand plotted as mean values of five to thirteen experiments ± SD. (E) Gap-filling activity 743 \nassessed in HEK-293T cells knocked-out (KO) for different NHEJ genes, as indicated. Results 744 \nare normalized to the NHEJ-proficient parental HEK-293T cell line and plotted as mean values 745 \n \n \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 36 \nof four to five experiments ± SD. Inset: control western blot on whole cell protein extracts from 746 \nthe different KO cells. (F) DNA junction analysis following gap -filling assay. Left: agarose 747 \ngel electrophoresis showing PCR products amplified around the junction following stable 748 \ngenome integration of the reporter substrate and gap -filling reaction (see the Materials and 749 \nmethods section). Three positive red -fluorescent clones (R1, R2 and R3) and three non -750 \nfluorescent clones as a control (C1, C2 and C3) were analyzed. Expected length of the PCR 751 \nfragments are indicated. Right: the DNA sequences of the PCR fragments are aligned with the 752 \nreporter substrate sequence using the Snapgene ® software (Dotmatics). (G) Western blot on 753 \nwhole cell protein extracts from HEK-293T cells knocked-out (KO) for POLL, POLM or both 754 \ngenes. When indicated, POLL KO cells were complemented with expression of ectopic WT or 755 \ncatalytic dead Pol . (H) Gap-filling activity assessed in HEK -293T cells knocked -out (KO) 756 \nfor POLL, POLM or both genes. Results are normalized to the parental HEK -293T cell line 757 \nand plotted as mean values of four to fourteen experiments ± SD. P -values from Student’s t -758 \ntest between the indicated conditions are as follows: 293T versus KO PolL (<0.0001 ****), 759 \n293T versus KO PolL + Poll -WT (<0.0001 ****), 293T versus KO PolM (0.0002 ***), KO 760 \nPolL versus KO PolL + Poll -dead (<0.0001 ****), KO PolL+PolM versus KO PolL + Poll -761 \ndead (0.0001 ***). 762 \n 763 \n 764 \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint \n\n 37 \nFigure S8: (A) Western blot on whole cell protein extracts from HEK -293T cells knocked-out (KO) 765 \nfor POL and complemented with an empty vector (EV) or expression vectors for wild -type (WT) or 766 \nthe indicated mutants of Pol . (B) Western blot on whole cell protein extracts from HEK-293T/mAID-767 \nKu70 cells depleted of endogenous Ku70 in the presence of auxin (+IAA) and rescued with ectopic 768 \nexpression of either WT or mutated forms of Ku70, as indicated. Asterisk indicates the position of 769 \nmAID-Ku70 signal just below that of Ku80, which persisted after previous hybridization of the 770 \nmembrane with anti -Ku70 antibody. (C) Gap-filling activity (red bars) or direct end -joining activity 771 \n(orange bars) assessed in parallel in HEK -293T cells knocked-out for POLL and complemented with 772 \nectopic expression of wild-type (WT) or different mutants of Pol . Results are normalized to the WT 773 \ncondition and plotted as mean values of four to five experiments ± SD. P -values from Student’s t-test 774 \nbetween the considered mutants, for gap -filling and direct end -joining activities, respectively, are as 775 \nfollows: Pol -dead versus Poll -R57E (<0.0001 ****; 0.2862 ns), Poll -dead versus Poll -R57E-dead 776 \n(<0.0001 ****; 0.0587 ns). (D) HEK-293T/mAID-Ku70 cells were seeded in 12-well plates and treated 777 \nwith auxin (+IAA) for the indicated time. Cell proliferation was then analyzed continuously up to 7 778 \ndays by assessing confluence with an IncuCyte-ZOOM (Essen Bioscience). 779 \n 780 \n 781 \n 782 \nReferences 783 \n1. Zhao, B., Rothenberg, E., Ramsden, D.A. & Lieber, M.R. The molecular basis and disease 784 \nrelevance of non-homologous DNA end joining. Nat Rev Mol Cell Biol 21, 765-781 (2020). 785 \n2. Seif-El-Dahan, M. et al. PAXX binding to the NHEJ machinery explains functional redundancy 786 \nwith XLF. Sci Adv 9, eadg2834 (2023). 787 \n3. Tang, J. et al. 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Hot Fusion: an efficient 864 \nmethod to clone multiple DNA fragments as well as inverted repeats without ligase. PLoS One 865 \n9, e115318 (2014). 866 \n 867 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 13, 2024. ; https://doi.org/10.1101/2024.08.12.607588doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}