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C-terminal region of bacterial Ku controls DNA bridging, DNA threading and recruitment of DNA ligase D for double strand breaks repair

Abstract Non-homologous end joining is a ligation process repairing DNA double strand breaks in eukaryotes and many prokaryotes. The ring structured eukaryotic Ku binds DNA ends and recruits other factors which can access DNA ends through the threading of Ku inward the DNA, making this protein a key ingredient for the scaffolding of the NHEJ machinery. However, this threading ability seems unevenly conserved among bacterial Ku. As bacterial Ku differ mainly by their C-terminus, we evaluate the role of this region in the loading and the threading abilities of Bacillus subtilis Ku and the stimulation of the DNA ligase LigD. We identify two distinct sub-regions: a ubiquitous minimal C-terminal region and a frequent basic C-terminal extension. We show that truncation of one or both of these sub-regions in Bacillus subtilis Ku impairs the stimulation of the LigD end joining activity in vitro. We further demonstrate that the minimal C-terminus is required for the Ku-LigD interaction, whereas the basic extension controls the threading and DNA bridging abilities of Ku. We propose that the Ku basic C-terminal extension increases the concentration of Ku near DNA ends, favoring the recruitment of LigD at the break, thanks to the minimal C-terminal sub-region.


INTRODUCTION
The genome of each cell is constantly exposed to a variety of damages caused by endogenous or exogenous factors (1). DNA double strand break (DSB) is a lethal DNA lesion as a single unrepaired DSB is sufficient to trigger cell death (2) and DSB misrepair can lead to chromosome rearrangements, a well-recognized cause of cancerogenesis in mammalian cells (3,4). To survive such deleterious event, cells have evolved two main DSB repair pathways: Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ).
The NHEJ pathway is a ligation process involving core and accessory NHEJ factors (5). The core factors are the Ku protein and a specific DNA ligase. In eukaryotes, the Ku protein is a heterodimer formed by the association of the Ku70 and Ku80 subunits; the ligation complex is constituted by XLF/Cernunnos, XRCC4 and DNA ligase IV. Other factors are recruited to form the complete and active eukaryotic NHEJ nucleoprotein complex which can process DNA ends even if broken ends cannot directly be joined by the DNA ligase complex (DNA-PKcs, Artemis, PNK and and X-family polymerases (5)). In prokaryotes, the core NHEJ factors are composed of the homodimeric Ku and LigD proteins (6)(7)(8)(9). LigD displays a ligase activity that is often associated with additional polymerase and/or nuclease activities (8,10,11). Other Ku partners, so far identified in mycobacteria only, come into play when the main NHEJ pathway is partially deficient (12,13) or to achieve yet undefined roles (14,15).
The eukaryotic Ku displays multiple roles in the NHEJ pathway. Ku is the first NHEJ protein recruited to the DNA broken ends. Ku protects DNA ends from nuclease activities and is involved in the regulation of the DSB repair pathway choice in eukaryotes (4). The human Ku displays a 5 -dRP lyase activity (16) showing that Ku participates also in the DNA repair process; this lyase activity was also characterized in two bacterial Ku proteins (17). Finally, Ku proteins are required as a scaffold for the assembly on the DSBs In most bacterial Ku, this minimal Cter is further extended by a variable sequence that tends to be enriched in basic amino acid residues. We refer to this region as the extended Cter. We purified and characterized the biochemical properties of Ku Bsub truncated of the extended Cter or the complete C-terminal region. Both the minimal and the extended Cter are involved in the Ku dependent stimulation of DSB repair by LigD Bsub . However, these two sub-regions have also distinct roles. The conserved minimal Cter is required for the interaction between Ku Bsub and LigD Bsub . The extended Cter is involved in the interaction with DNA independently on the presence of DNA ends, promotes DNA bridging and regulates the threading of Ku Bsub inward the DNA molecule following DSBs ends binding.

Bacterial strains and plasmids
Escherichia coli strains used were MC1061 (31) for plasmid constructions and ER2566 from NEB for protein expression. The sequences of all the primers used in this study are available upon request.
The Ku Bsub protein used in this study had the same sequence than the annotated Ku protein from B. subtilis 168 (NP 389224.1) from the methionine at position 18 to the serine at position 311 (amino acids positions of the annotated NP 389224.1, see Results) and was tagged by a hexahistidyl-tag (His 6 ) at its N-terminus. The Ku Bsub protein was produced from an E. coli expression-optimized Open Reading Frame (ORF) created by DNA2.0 (Menlo Park, CA, USA) which was derived from the ykoV (named hereafter ku) ORF in order to decrease mRNA secondary structures and rare codons patches in E. coli without changing the amino-acid sequence of the protein. The optimized ORF was flanked by the coding sequence of a His 6 -tag followed by a NdeI site on its 5 side, and by a XhoI site on its 3 side. The resulting ORF was cloned under the T7 promoter of the pJ411 vector (DNA2.0) giving the pSMG249 plasmid.
The ORFs coding for Ku exCter and Ku core mutant proteins (corresponding to the M18 to P277 (Ku exCter) or M18 to P256 (Ku core) sequence of the NP 389224.1 protein, see Figure 1) were amplified from the pSMG249. The NdeI-XhoI fragment of pSMG249 encompassing the ku gene was replaced by the PCR products, giving the plasmid pSMG219 and pSMG224 coding for a N-terminal His 6tagged Ku exCter and a N-terminal His 6 -tagged Ku core proteins, respectively (named hereafter Ku exCter and Ku core).
The B. subtilis ykoU gene coding for the LigD protein was modified by DNA2.0 to optimize its expression in E. coli. The modified gene was flanked by a NdeI site on the 5 side and by the coding sequence of a His 6 -tag followed by a XhoI site on its 3 side. The cloning of this modified gene in the pJ411, giving the plasmid pSMG217, allowed the expression of the recombinant C-terminal His 6 -tagged LigD protein (named hereafter LigD Bsub ).
The plasmid pSMG240 allowing expression of the MRGS(H) 6 GS-tagged Green Fluorescent Protein fused to the basic extended Cter sub-region of Ku Bsub (named hereafter GFP-KC, the extended Cter part of Ku correspond-ing to the sequence from P277 to S311 of the NP 389224.1 annotated protein) was obtained in two steps. Firstly, the corresponding 3 part of the ku gene was amplified from pSMG249, using primers allowing flanking of the PCR product by ApaI and XhoI sites on the 5 and 3 sides, respectively. This 3 part of ku was cloned in frame at the 3 of the GFP gene of the plasmid pSG1729 (32) cut by ApaI and XhoI, giving rise to the plasmid pSMG239. Secondly, GFP-KC gene was amplified from pSMG239 using primers allowing its integration between BamHI and XhoI sites of plasmid pQE9 (Qiagen).
The MRGS(H) 6 GS-tagged GFP (named hereafter GFP) protein was obtained via the expression of the corresponding gene on the pSMG245. This plasmid was constructed directly by amplification of the GFP gene on the plasmid pSG1729 and its integration between the BamHI and XhoI sites of the pQE9 plasmid.

Comparative sequence analysis
Profile HMMs are considered as one of the most powerful methods to identify remote protein homologues (PMID:9837738). We used profile HMMs to build a list of Ku proteins found in the 2645 complete prokaryotic genomes available in September 2013 as described below.
The first task consisted in collecting from Genbank a non-redundant and diverse set of sequences annotated as corresponding to Ku proteins that could be used to learn the parameters of the profile HMM. We first retrieved from Genbank the 1048 protein sequences corresponding to protein coding genes that have been annotated as belonging to Cog1273. This data set was subjected to an allagainst-all comparison with blastp 2.2.26 (PMID:9254694). This allowed to exclude a small subset of 16 highly atypical sequences that displayed hits (E-value cut-off 0.00001) with less than half of the 1048 protein sequences (this included 6 proteins from the eukaryote Leishmania, 2 proteins from bacterium Rhodanobacter fulvus without similarity and whose annotation was considered as unsure, and 8 protein fragments of length less than 100 amino acids). The level of protein sequence similarity between a pair of proteins was summarized by the raw Blast Score Ratio defined as BSR[P1,P2] = BS[P1,P2] / max{BS[P1,P1],BS[P2,P2]}, where P1 and P2 are two proteins and BS[P1,P2] is the Blast raw score between P1 and P2 (PMID:15634352). Out of the remaining 1032 protein sequences we used a simple greedy algorithm to select a subset of 89 sequences representatives of the whole diversity defined such that any of the 1032 protein sequences has at least one representative in the subset with BSR ≥ 0. 5  Using HMMER 3.0 (PMID:22039361), the 71 full-length Ku domains realigned with MUSCLE served to build an HMM profile that was used to query the complete genomes. Our final list of 689 Ku proteins consisted of all the hits scoring above 50% of the highest score (E-values were highly significant <10 −47 for all those hits). The choice of this score cut-off reflected a marked discontinuity in the distribution of the scores: no hits were found between 54% and 23% of the highest score and only 13 hits with E-value < 0.05 were found below the chosen score cut-off.
The 689 Ku proteins identified in complete genomes were aligned with MUSCLE. The Ku core domain as well as the minimal and extended Cter regions were delineated based on this alignment. Isoelectric points (pI) were determined with Bioperl using EMBOSS amino acid pK values (PMID:12368254, PMID:10827456). A tentative phylogenetic tree was obtained from the multiple alignment with the maximum likelihood algorithm implemented in Phyml v3.0 with JTT model of protein evolution and a gamma distribution (approximated with 4 categories of sites) to account for rate heterogeneity (PMID:20525638). The graphical representation of the tree was drawn with R package ape (PMID:14734327).

Purification of the proteins produced in E. coli
Ku Bsub and C-terminal truncated mutants. E. coli ER2566 cells transformed with pSMG249 (producing Ku Bsub ), pSMG219 (producing Ku exCter) or pSMG224 (producing Ku core) were grown at 30 • C in 2 liters of LB medium supplemented with 25 mg/ml thymine and 30 g/ml kanamycin to A 600nm = 0.7. Production of proteins was induced with 0.5 mM IPTG for 3 h at 30 • C. Cells were harvested by centrifugation and pellets were resuspended in 40 ml lysis buffer (50 mM Tris-HCl pH 8, 0.2 M NaCl, 0.1% Triton, 0.1% Nonidet P40, 0.2 mg/ml lysozyme) and incubated 1 h at 20 • C. Cells were broken by a freeze/thaw step in liquid N 2 /37 • C. All subsequent steps were carried at 4 • C. Two milliliters of polyethyleneimine (PEI) 10%, pH 8 was added to precipitate nucleic acids; the mixture was stirred for 1 h and then centrifuged 1 h at 100 000 g. Soluble phase was next precipitated with raising concentrations of ammonium sulfate (AS) by steps of 10% of saturation, and AS pellets were resuspended with 25 ml buffer P 1 (50 mM Tris-HCl, pH 8, 1 M NaCl). Fractions were checked for the presence of the protein of interest by 10% SDS-PAGE. Ku Bsub was mainly found in the pellet obtained at 50% of AS saturation, Ku exCter and Ku core in the pellet obtained at 40% of AS saturation. The AS pellets were resuspended with 40 mL of buffer P 1 and were loaded onto a Ni 2+ affinity column (Ni-NTA agarose, QIAGEN) pre-equilibrated in buffer P 1 . The Ni-NTA column was washed with the same P 1 buffer then with P 1 buffer supplemented with 20 mM imidazole. Ku Bsub and C-terminal mutants were eluted in buffer P 1 supplemented with 250 mM imidazole and dialysed against buffer S (50 mM Tris-HCl pH 8, 0.15 M NaCl, 5% glycerol) for storage at −80 • C before their use in small angle X-ray scattering (SAXS) analyses or against buffer D (50 mM Tris-HCl pH 8, 0.4 M NaCl, 50% glycerol, 1 mM DTT) before their storage at −20 • C for all other experiments.
LigD Bsub protein. E. coli ER2566 cells transformed with plasmid pSMG217 (producing LigD Bsub ) were grown at 30 • C in 2 liters of LB medium containing 25 mg/ml thymine and 30 g/ml kanamycin. At A 600nm = 0.7, culture was shifted at 16 • C before induction with 0.5 mM IPTG for 18 h. Cells were harvested by centrifugation and the pellet was resuspended in 40 ml of P 1 buffer and stored at −20 • C. All subsequent steps were carried at 4 • C. Cells were lysed by sonication and centrifuged at 100 000 g for 1 h. AS was added to the lysis supernatant up to 30% of AS saturation. The AS pellet was resuspended in 40 ml of P 1 buffer and was loaded onto a Ni 2+ affinity column pre-equilibrated in buffer P 1 . The Ni-NTA column was washed with P 1 buffer and then with P 1 supplemented with 20 mM imidazole. The protein was eluted with the same buffer supplemented with 250 mM imidazole and was dialyzed against buffer D. LigD Bsub was diluted with buffer P 0 (50 mM Tris-HCl, pH 8) to reduce the NaCl concentration to 75 mM and loaded onto a 1 ml Hitrap Heparin (GE Healthcare). LigD Bsub was eluted in one step in buffer P 1 . The protein was then dialyzed against buffer D prior storage at −20 • C.
GFP and GFP-KC proteins. The GFP was produced from plasmid pSMG245 in E. coli ER2566 cells grown at 30 • C in 2 liters of LB medium containing 25 mg/ml thymine and 100 g/ml ampicillin to A 600nm = 0.7 and induced 18 h with 0.5 mM IPTG at 16 • C. After centrifugation, harvested cells were resuspended in 40 ml of buffer P 1 , lysed by sonication and centrifuged at 100 000 g for 1 h at 4 • C. All subsequent steps were carried at this temperature. Supernatant was loaded onto a Ni 2+ affinity column pre-equilibrated in buffer P 1 . The Ni-NTA column was washed with buffer P 2 (50 mM Tris-HCl, pH 8, 2 M NaCl) supplemented with 25 mM imidazole. The protein was eluted with buffer P 2 supplemented with 250 mM imidazole and dialysed against buffer D prior storage at −20 • C.
The GFP-KC protein was produced from a freshly pSMG240 transformed colony of E. coli ER2566. Its purification was done as described for the GFP, except that a supplemental purification step was done. The dialyzed protein of the Ni-NTA eluate was diluted with buffer P 0 to reduce the NaCl concentration to 50 mM, loaded onto a cation exchange Hitrap SP 1 ml column (GE Healthcare) equilibrated with buffer P 0.05 (50 mM Tris-HCl, pH 8, 50 mM NaCl), and eluted with a linear 15 ml gradient of NaCl from 50 mM to 1 M in buffer P 0 . The GFP-KC obtained was then dialysed against buffer D and stored at −20 • C.
All these proteins were obtained at 95% purity and their concentration was determined by UV absorption at = 280 nm on a NanoDrop 1000 Spectrophotometer (Thermo Scientific).

SAXS data collections
Ku Bsub and the two C-terminal truncated mutants were analyzed by SAXS experiments to describe their structural organization. All protein solutions were centrifuged for 10 min at 10 000 g prior to X-ray analysis in order to eliminate aggregates and their concentration was measured by UV. Protein samples were prepared at a final concentration of 95 M, stored at 4 • C and then directly used for the experiments.
SAXS experiments were conducted on the SWING beamline at the SOLEIL synchrotron ( = 1.033Å). The Aviex charge-coupled device detector was positioned to collect data in the Q-range 0.008-0.5Å −1 (Q = 4sin −1 , where 2 is the scattering angle). All solutions were mixed in a fixed-temperature (15 • C) quartz capillary with a diameter of 1.5 mm and a wall thickness of 10 m, positioned within a vacuum chamber. Fifty microliters of samples of proteins at 95 M were injected onto a size-exclusion column (SEC-3 300 Agilent), using an Agilent HPLC system, and eluted directly into the SAXS flow-through capillary cell at a flow rate of 0.2 ml/min (33). The elution buffer consisted of 50 mM Tris-HCl, pH 8, 150 mM NaCl and 2% glycerol. SAXS data were collected continuously, with a frame duration of 1.0 s and a dead time between frames of 0.5 s. Data reduction to absolute units, frame averaging and subtraction were done using FOXTROT, a dedicated home-made application. All subsequent data processing, analysis and modeling steps were carried out with PRIMUS and other programs of the ATSAS suite (34).
Data evaluation. The experimental SAXS data for all samples were linear in a Guinier plot of the low q region, indicating that the proteins did not undergo aggregation. The radius of gyration R g was derived by the Guinier approximation I(q) = I(0) exp(-q 2 R g 2 /3) for qR g < 1.0 using PRIMUS (35). Interference-free SAXS profiles were estimated by extrapolating the measured scattering curves to infinite dilution. The program GNOM (36) was used to compute the pair-distance distribution functions, p(r). This approach also features the maximum dimension of the macromolecule, D max ( Figure 3A and B). The Porod volume were determined with PRIMUS, the molecular weights were estimated with SAXS-MoW (37) and the Porod coefficients were calculated using SCATTER (38).
Ab Initio modeling. The overall shapes of the entire assemblies were restored from the experimental data using the program GASBOR (39). These models were averaged to determine common structural features and to select the most typical shapes using the programs DAMAVER (40) and SUPCOMB (41).

Molecular modeling.
Due to the absence of structure of Ku Bsub , the initial model building for a dimeric Ku Bsub was performed using the structure of the complex human Ku heterodimer-DNA (code pdb : 1JEY, (21)) as template. As previously described (19), the sequence of the Ku core domain of the Ku70 subunit has a better homology with the Ku core domain of the Ku Bsub sequence than the Ku80 subunit and the C-terminal domain of Ku70 and Ku Bsub present a strong sequence divergence. We generated a homodimer composed of two Ku70 deleted of the N-terminal (residues 34 to 260) and C-terminal regions (residues 506 to 534) defining a chimera Ku core domain without DNA, keeping the ring structure of the DNA recognition domain (see Supplementary Results, Figures S2 and S3).
To generate a structural model of the Ku Bsub and its derivative mutants compatible with the SAXS data, we per-Nucleic Acids Research, 2016, Vol. 44, No. 10 4789 formed atomic modeling using DADIMODO (42), a genetic algorithm based rigid-body refinement analysis program. The initial structures were first minimized by keeping fixed the main chain of the rigid domain. The declared flexible parts (C-terminal regions of the homodimeric Ku Bsub ) were then submitted to random mutations, whereby phi/psi angles were changed with a maximum amplitude of 45 • per step. Continuity of the structure was constrained by subsequent energy minimization. A SAXS 2 value was then computed for each eligible structure, using CRYSOL (43) with 50 harmonics. The selection protocol, based on tournament method (42) with progressive increase of the selection, allows the final selection of the best fitting models (Figure 3). To avoid overfitting and to control validity of the models, a 2 free metric was also calculated using SCAT-TER (38).

DNA preparation
The 279 bp linear dsDNA molecules were obtained by PCR amplification of a region of the B. subtilis genome. The 1001 bp and 1876 bp dsDNA molecules were amplified from the pZE14 plasmid (44). PCRs were done using 5phosphorylated primers and the Phusion polymerase (Thermofisher) in order to generate blunt-ended products. For the DNA bridging assay, the one-end biotinylated 1001 bp dsDNA molecule was prepared using one 5 -biotinylated primer and one 5 -phosphorylated primer. Amplified DNA molecules were purified on agarose gel using the QIAquick PCR purification kit (QIAGEN).
The pUC19 plasmid (2686 bp) was isolated from E. coli DH5␣ cells using Isolating Recombinant Bacmid DNA protocol (Invitrogen) and purified on an ion exchange Resource Q column (GE Healthcare) with a chromatography SMART system (GE Healthcare). For electron microscopy experiments, all DNA molecules were purified on a MiniQ anion exchange column (GE Healthcare) with a chromatography SMART system. The purified DNA was precipitated and resuspended in a 10 mM Tris-HCl, pH 7.5, 1 mM EDTA buffer.

Ligation assay
One hundred nanograms (∼150 fmol) of the 1001 bp linear dsDNA molecules with 5 -phosphorylated ends were preincubated with 9 pmol of Ku Bsub or C-terminal Ku truncated mutants in 6.52 l of buffer L (50 mM Tris-HCl pH 8, 1 mM DTT, 5 mM MgCl 2 , 30 mM NaCl and 3% glycerol) for 30 min at 37 • C. Serial dilutions of the LigD Bsub protein in buffer L supplemented with 0.39 mM adenosine triphosphate (ATP) were prepared and 11.48 l of each dilution were added to the reaction (allowing final concentrations of LigD Bsub indicated in the legend of Figure 2). The final volume of each reaction mixture was 18 l in buffer L supplemented with ATP (0.25 mM final concentration). Ligation reactions were stopped after 2 h at 37 • C by adding 2 l of stop buffer (5 mg/mL proteinase K, 2% SDS and 0.1 M EDTA) and incubating at 50 • C for 30 min. A total of 6.7 l of DNA loading buffer (33% glycerol and 0.25% xylene cyanol) were added, reactions were cooled on ice, centrifuged 1 min at 14 000 g and half of the reaction volume was loaded onto a 0.8% agarose gel in Tris-Borate-EDTA (TBE) buffer. After electrophoresis, DNA was stained by the SYBR Gold reagent (Invitrogen) and the gel was photographed using a Chemidoc apparatus (Bio-Rad Laboratories). Quantification of the unligated DNA substrate was done using the Image Lab v.5.0 software (Bio-Rad Laboratories).

Gel filtration assay
Five nmol of Ku Bsub , Ku exCter or Ku core proteins, 2.5 nmol of LigD Bsub or mixes of 2.5 nmol of LigD Bsub with 5 nmol of Ku Bsub or C-terminal truncated mutants were incubated in 600 l of buffer P 0.15 (50 mM Tris-HCl, pH 8, 150 mM NaCl) for 20 min at 30 • C. After 5 min of centrifugation at 13 000 g, the soluble fraction was injected on a Superdex 200 10/300 GL (GE Healthcare) equilibrated with buffer P 0.15 . Twenty two fractions of 500 l were collected, ranging from approximately 700 kDa (the void volume) to 10 kDa. To compare the elution volumes of the proteins, 5 l of SDS-loading buffer were added to 20 l of each of these fractions and loaded onto 10% acrylamide SDS-PAGE gels. After migration, gels were stained with Coomassie blue.

Electrophoretic mobility shift assay (EMSA)
Various amounts of proteins were added to 50 ng of DNA substrate in 20 l reaction mixtures in buffer L. After incubation at 30 • C for 20 min, reactions were mixed with 4 l of EMSA loading buffer (50% glycerol, 0.04% xylene cyanol) prior loading on a 5% native polyacrylamide gel (29:1, in Figure 6A) or on a 0.7% GTG-agarose (Lonza) gel in TBE buffer ( Figure 5 panels A and F), and then migrated at 4 • C at 15 V/cm for 2 h. Following electrophoresis, gels were colored with SYBR-Gold and revealed on a Chemidoc apparatus. Quantification of unbound linear dsDNA molecules was done using the Image Lab v.5.0 software (Bio-Rad Laboratories).

Transmission electron microscopy (TEM) analysis
TEM samples preparations were performed by positive staining as previously described (45). All nucleoprotein complexes were formed in 10 mM Tris-HCl pH 8, 50 mM NaCl or 10 mM Tris-HCl pH 8, 200 mM NaCl binding buffer with 0.3 nM DNA substrates and in presence of various concentrations of Ku proteins. Five microliters of DNA-protein reaction were deposited onto a 600 mesh copper grid coated with a thin carbon film, previously activated by glow-discharge in the presence of pentylamin (Merck, France). Grids were washed with aqueous 2% (w/vol) uranyl acetate (Merck, France) and then dried with ashless filter paper (VWR, France). TEM observations were carried out on a Zeiss 912AB transmission electron microscope in filtered crystallographic dark field mode. Electron micrographs were obtained using a ProScan 1024 HSC digital camera and iTEM software (Olympus, Soft Imaging Solutions).
To characterize the different classes of DNA molecules bound by Ku Bsub or by C-terminal mutants, the number of naked DNA molecules and DNA-protein complexes not trapped into networks or aggregates per m 2 was determined at different incubation times. This allowed (i) to calculate the frequencies of these untrapped DNA molecules (and consequently frequencies of networks and aggregates, Figure 8D) and (ii) to estimate the proportion of the different classes of untrapped DNA molecules ( Figure 8E). The nucleoprotein complexes untrapped into networks or aggregates were divided in four classes: naked DNA, complexes with proteins at one or both DNA ends and complexes with proteins inside the DNA molecule and one, two or without covered ends (see Figure 8E). The number of molecules in each class is counted, normalized at 100 and represented by a histogram.
To compare the DNA threading ability of the proteins, the DNA length covered by each protein and the total contour length are measured for each nucleoprotein complex untrapped into networks using the iTEM software. The percentage of DNA covered by each protein corresponds to the ratio of the sum of DNA length covered by Ku Bsub or truncated mutants at each end over the total length of the DNA molecule. The distribution is represented by box and whisker plots and was statistically analyzed with a Mann-Whitney U test done with Prism5 software (Graphpad software).

DNA bridging assay
This assay was derived from the DNA bridging assay described in (46). Ten microliters of Streptavidin-coated Dynabeads Kilobase BINDER (Invitrogen) were washed three times with 40 l of reaction buffer (10 mM Tris-HCl, pH 8, 50 mM NaCl and 400 g/ml BSA) and resuspended in 40 l of reaction buffer. Two hundred nanograms of the blunt-ended 1001 bp PCR product either 5 -phosphorylated on both ends (named hereafter 1001PP, control experiment) or 5 -biotinylated on one end and 5 -phosphorylated on the other end (1001BP) were incubated for 5 min with the beads. Beads were washed twice with 40 l of reaction buffer, resuspended in 36 l of the same buffer and 4 l of 40 M Ku Bsub , Ku exCter or Ku core solutions were added. After 20 min incubation at 30 • C, beads were washed twice with 40 l of reaction buffer and resuspended in 60 l of the same buffer. At this step, a 20 l aliquot was analyzed by SDS-PAGE to check fixation of the proteins (named 'M' fraction in Figure 7A).
Next, 200 ng of the blunt-ended 1876 bp PCR product (5 -phosphorylated at both ends) were added to the remaining 40 l beads suspension and incubated for 5 min at 30 • C. Beads were washed twice with 40 l of the reaction buffer without BSA and then resuspended in 40 l of the same buffer.
To evaluate the amount of captured 1876 bp fragment, 20 l of the beads suspension were treated with 2 l of stop buffer for 30 min at 50 • C and then resolved by electrophoresis on a 0.7% TBE agarose gel. DNA was stained by the SYBR Gold reagent and the gel was photographed using a Chemidoc apparatus. The remaining 20 l of the bead suspension (named 'E' fraction in Figure 7A) were analyzed by SDS-PAGE.

Nuclease protection assays
The 1001 bp linear dsDNA molecule used in the T5 exonuclease assay was the same as those used in the ligation assay described in the Materials and Method. One hundred nanograms of this 5 -phosphorylated blunt-ended molecule was preincubated with Ku Bsub or Ku exCter proteins in Buffer L (50 mM Tris-HCl pH 8, 1 mM DTT, 5 mM MgCl 2 , 30 mM NaCl and 3% glycerol) for 30 min at 37 • C. A total of 0.5 U of T5 exonuclease (Biolabs) was added to the reaction mixture (final volume: 18 l) and reactions were incubated for 1 h at 37 • C. Reactions were stopped by adding 2 l of stop buffer (5 mg/mL proteinase K, 2% SDS and 0.1 M EDTA) and incubating at 50 • C for 30 min. Then 6.7 l of loading buffer (33% glycerol and 0.25% xylene cyanol) were added to the reaction. Degradation products corresponding to the half of the reaction were resolved by electrophoresis on a 0.8% agarose gel in TBE buffer. DNA was stained by SYBR Gold and the gel was photographed under UV light.
A HincII endonuclease assay was performed to test the Ku mediated protection of DNA against restriction. We used the same blunt-ended 1001 bp linear dsDNA molecule with 5 phosphorylated ends. This molecule contains two HincII sites (see Figure 9). The assay was the same as the T5 exonuclease assay described above except that 50 ng of the 1001 bp substrate was used and the digestion reaction were done in buffer L supplemented with 0.1 mg/ml BSA and the incubation time with HincII (4.8 U) was 2 h at 37 • C. Digested products present in 20 l out of the 26.7 l of the final reaction volume were analyzed. After SYBR Gold staining, the gel (1% agarose in TBE) was photographed using a Chemidoc apparatus. Quantification of digested products was done using the Image Lab v.5.0 software (Bio-Rad Laboratories).
Kinetics of HincII digestion of the 1001 bp dsDNA substrate preincubated with different Ku concentrations was done as described for the HincII endonuclease assay except that 400 ng of the 1001 bp substrate and 3.84 U of HincII were used in a final reaction volume of 144 l. Samples of 18 l were extracted at different time of the incubation at 37 • C and analyzed as described above.

Prokaryotic Ku core domain is associated with a conserved minimal Cter region frequently extended by a basic tail
To examine the domain architecture of prokaryotic Ku proteins, we searched 2645 complete prokaryotic genomes for genes encoding putative Ku proteins (see Materials and Methods), and we identified 689 ku genes. 20.2% of all genomes contain a unique ku gene (528 bacterial genomes and only 7 euryarchaeotal genomes) and 3.5% of genomes contain more than one ku (details in Supplementary Table  S1). For instance, 4 ku homologues are found in the Sinorhizobium meliloti genome (47), with some bacteria of the Rhizobium genus having 6 ku genes. The microorganisms bearing a ku gene belong predominantly (518/528) to the Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes bacterial phyla ( Figure 1A). However, even in these phyla, ku genes were found only in a fraction of the sequenced genomes: 54.7% of the Actinobacteria, 23.6% of the Fir- Alignments of amino acid sequences were done using the multiple sequence alignment Clustal Omega method (59) using the default value for each parameter. The conserved Ku core and minimal Cter domains are highlighted in gray and blue respectively. The common extended Cter is highlighted in yellow. Conserved residues between Ku proteins are indicated by an asterisk. Strongly and weakly similar residues are indicated by a column and a period respectively. A hyphen is used to represent a gap in the sequence. The secondary structure prediction (se. stru.) of the Ku Bsub from the first methionine of the Ku core domain to the end of the protein was obtained using the GOR version IV method (60). c, e and h stand for random coil, extended strand and alpha helix, respectively. Arrows indicate last residues of the Ku core (proline pointed in the minimal Cter) and the Ku exCter mutants (proline pointed in the extended Cter). micutes, 19.9% of the Proteobacteria and 14.4% of the Bacteroidetes (Supplementary Table S2).
All prokaryotic Ku proteins contain a central Ku core domain ((19), in gray in Figure 1D) displaying high level of homology with the eukaryotic Ku70 proteins and ranging from 187 and 311 amino acid residues (average 236) in length. The Ku core domain is generally very close to the Nterminus as 92.9% of Ku proteins have an N-terminal extension of 5 residues or less ( Figure 1D and Supplementary Table S1). Extending the Ku core domain, all prokaryotic Ku proteins display a C-terminal region ranging from 19 to 173 residues (average 59) in length. Sequence alignments delineated two distinct domains in the bacterial Ku C-terminal region. Directly adjacent to the Ku core, a small domain of 12 to 28 residues, hereafter named minimal Cter domain, exhibits some similarity across prokaryotic Ku proteins ( Figure 1D). The minimal Cter domain is followed by an extended C-terminal domain (hereafter named extended Cter) in the vast majority of prokaryotic Ku proteins (Figure 1C). The extended Cter domain varies considerably in length, ranging from 1 to 155 residues. A remarkable feature of this domain is that it tends to be markedly basic ( Figure 1B), with a mean isoelectric point of 11.4 relative to 5.5 for the Ku core and the minimal Cter domains. This is due to a bias in the amino acid composition (lysine and to a lesser extent arginine residues), already noticed for Ku Paer and Ku Msme (7,30). The extended Cter regions do not share any significant sequence similarity ( Figure 1D).

Purification of B. subtilis Ku protein variants of the Cterminal region
The B. subtilis Ku protein (NP 389224.1) is composed of a 236 residues long Ku core domain with an unusually long (17 residues) N-terminal domain, a minimal Cter domain (18 residues) and an extended Cter domain (40 residues) comprising 11 lysine and arginine residues (isoelectric point 11.7, Figure 1D). The transcription start site (TSS) of the ykoV gene encoding Ku Bsub was mapped in a large scale structural reannotation of the B. subtilis genome (48). The ykoUV operon, which is under the control of the SigG transcription factor, is located downstream of the predicted start codon in UniprotKB, indicating an annotation error. An ATG located 17 codons downstream and corresponding to the first position of the Ku core domain is the likely Ku start site as it is compatible with the mapped TSS (U1083) and consistent with conserved Ku core domain ( Figure 1D).
We cloned, expressed in E. coli and purified LigD Bsub , Ku Bsub and two C-terminally truncated Ku mutants. Ku Bsub was truncated at two sites (indicated by arrows in Figure  1D) to eliminate the extended Cter domain (Ku exCter) or the entire C-terminal domain (Ku core). The biochemical properties of the purified full length Ku Bsub and truncated mutants proteins were compared for interaction with LigD Bsub and stimulation of its activity and for interaction with various DNA substrates.

The C-terminal domain of Ku Bsub is required to stimulate the ligase activity of LigD Bsub
The primary function of Ku in bacterial NHEJ is to recruit LigD to the DNA ends thereby stimulating LigD end join-ing activity. We tested the stimulation of LigD Bsub end joining activity by Ku Bsub using the full length and C-terminally truncated Ku exCter and Ku core proteins. The Ku Bsub protein variants were preincubated with a 1001 bp long linear DNA substrate carrying 5 -phosphorylated blunt ends, at a 30-fold molar excess of Ku Bsub compared to DNA ends. Different amounts of LigD Bsub were added to the reaction mixture at a molar ratio ranging from 0.8-to 100-fold relative to DNA ends, and end joining DNA products were analyzed after 2 h incubation. Controls with Ku proteins only did not show formation of ligation products ( Figure  2A), indicating that purified Ku proteins do not display any end joining activities. High concentration of LigD Bsub exhibited a weak end joining activity. In sharp contrast, larger amounts of multimers of the DNA substrate were formed when both Ku Bsub and LigD Bsub proteins were present (Figure 2B), showing that Ku Bsub stimulates the ligase activity of the LigD Bsub , in agreement with a previous report (17). Increase of LigD Bsub concentration yielded DNA end joining products of increasing molecular weight, with a maximal ligation activity observed at 105 nM LigD Bsub corresponding to a ratio of 6.2 relative to DNA ends and 0.21 relative to Ku proteins. Further increase in LigD Bsub concentration resulted in a plateau in the ligation efficiency from 105 to 421 nM followed by a slight decrease ( Figure 2E). Similarly, the Ku exCter mutant protein stimulated LigD Bsub DNA end joining activity (Figure 2 panel C). However, the maximal stimulation required 8-fold higher LigD Bsub amount (842 nM) with Ku exCter than with Ku Bsub ( Figure 2E and compare panels B and C). This result implies that the basic extended Cter region of Ku Bsub plays a role in the Kudependent stimulation of the DNA end joining activity of LigD Bsub . In contrast, the Ku core mutant protein was unable to stimulate LigD Bsub DNA end joining activity ( Figure  2D).

C-terminal truncations of Ku Bsub do not affect oligomerization and global architecture of the Ku core domain
To understand why Ku C-terminal truncations alter the capacity of Ku Bsub to stimulate the ligase activity of LigD Bsub , we first analyzed the structural shape of the truncated mutants and full length Ku proteins by SAXS. The different proteins were injected to a size exclusion column and eluted directly into a SAXS flow through capillary cell. SAXS experimental data were recorded from q = 0.01Å −1 to q = 0.4 A −1 ( Figure 3A). As illustrated in Supplementary Figure  S1 panel A, the Guinier extrapolations did not show aggregation phenomenons and gave unambiguous values of Rg for Ku Bsub equal to 42.2Å, for Ku exCter = 34.2Å and Ku core = 28.4Å. The autocorrelation functions p(r) were calculated from the SAXS data ( Figure 3A, insert) giving information on the shape of the proteins, the maximum distance D max and the Rg values, similar to the ones calculated from the Guinier extrapolations ( Figure 3B). The p(r) of the Ku core had a bell shape corresponding to a globular protein whereas the two other constructs Ku exCter and Ku Bsub presented a low decrease of the shape for the high distances. This increase of the D max for the two other constructs (Ku exCter = 150Å and Ku Bsub = 190Å compared to Ku core = 90Å, Figure 3B) suggested that these proteins harbors an elongated part in the C-terminal region. The calculation of the Porod volume and the molecular weight confirmed that this increase of D max was due to an elongated part and not to a change of oligomeric state. This observation was confirmed by the determination of the Porod coefficient ( Figure 3B) and the normalized Kratky representation (Supplementary Figure S1B). For a globular form such as Ku core, the value of Porod coefficient is equal to 3 and the Kratky plot gives a plateau at high angles close to zero, whereas the loss of the sphericity due to the presence of the unfolded C-terminus region in Ku Bsub and Ku exCter de-creases the Porod coefficient and increases the height of the plateau at high angles on the Kratky plot.
Molecular modeling from these SAXS data required to assess the oligomerization state of the different proteins. To this end, proteins were cross-linked or not with Sulfo-EGS and analyzed by SDS-PAGE (Supplementary Figure S4). In the absence of the cross-linker, monomeric Ku Bsub (calculated molecular weight: 34 kDa), Ku exCter (30.6 kDa) and Ku core (28.2 kDa) proteins migrated with an apparent molecular weight of ∼37 kDa, 32 kDa and 28 kDa, respectively. Addition of Sulfo-EGS led mostly to the production of proteins migrating with apparent molecular weights of 80 kDa (Ku Bsub ), 70 kDa (Ku exCter) and 60 kDa (Ku core). These estimations are in good agreement with the calculated molecular weight of dimers and with biophysical parameters extracted from the SAXS data for these proteins ( Figure  3B). Therefore, truncation of the C-terminal domain does not alter the dimeric state of Ku Bsub .
Using scattering curves, low resolution structures of these proteins were calculated by ab initio modeling with GAS-BOR program by imposing a P2 symmetry (the choice for the P2 symmetry and comparison with low resolution structures using P1 symmetry is described in the Supplementary Results and Figure S2A). Selection of the most typical shape for each of the three Ku proteins is presented in Figure 3D (transparency spheres). The full length Ku Bsub protein is composed of a central globular domain displaying a ring shape which is extended by two arms on opposite sides and excluded from the ring shape domain (Figure 3D, left panel). This ring shape domain resembles to the three dimensional (3D) structure of the human Ku70/80 heterodimer (21) where the Ku core domain of the two proteins forms a ring able to encircle a dsDNA molecule (Supplementary Figure S2B). Interestingly, truncation of the extended Cter region of Ku Bsub led to a more globular protein with smaller arms and a maintained central globular domain ( Figure 3D, middle panel). The Ku core protein displayed only the ring shape with a dimension similar to those of the Ku Bsub and Ku exCter proteins ( Figure 3D, right panel).
Molecular modeling was performed using the known 3D structure of the human Ku protein (see Materials and Methods and Supplementary Results) and the collected SAXS data for Ku Bsub and C-terminal mutants. The theoretical SAXS curves produced from these calculated atomic models were compared to the experimental SAXS data ( Figure  3C). The low noisy data, the comparable values of 2 and 2 free values close to 1 and the quality of the residual plot show that the proposed molecular models are in agreement with the experimental SAXS data. Superimposition of the calculated atomic models ( Figure 3D, shown in cartoons) exhibited significant agreement in size and form with the ab initio calculated shapes (transparency spheres). A central ring shape can be modeled for the three proteins and extended arms are visible only for Ku Bsub and Ku exCter, arms that are longer in the case of the full length Ku protein. This ring shape domain fits also well with the 3D structure of the Ku core domain of the Ku70/80 heterodimer (Supplementary Figure S3). However, during the first molecular modeling process against SAXS data of the Ku core mutant, the modification of C-terminal region by the atomic modeling algorithm DADIMODO was not sufficient to accommodate SAXS data with a good agreement (see Materials and Methods), and the same phenomenon was observed with Ku Bsub and Ku exCter. A better fit was achieved by introducing flexibility in the DNA recognition region in a second cycle of molecular modeling. DADIMODO was allowed to modify the ring structure, initially folded in beta strand in the crystallographic structure (see the model of Ku Bsub in Figure 3D, shown in yellow). This modification does not affect the global structure, which always shows a LigD Bsub (2.5 nmol) were injected alone or together (as indicated on the right of the photographs) onto a gel-filtration column. A total of 20 l of eluted fractions (0.5 ml) were analyzed by SDS-PAGE. A photograph of each Coomassie blue stained gel is shown. Photographs are positioned so that eluted fractions collected at the same time after injection for each chromatography are vertically aligned. Molecular weight (in kDa) of standard proteins used to calibrate the column (arrow heads) or the gel (dashes) are indicated.
ring shape, but suggests that the region of DNA recognition could be flexible in solution when no DNA is docked inside.
From these structural modeling results, we propose that the conserved ring shape of the globular domain between the three proteins corresponds to the Ku core domain of Ku Bsub while the C-terminal domain is included in the arms.

Ku/LigD interaction requires the conserved minimal Cter region of Ku Bsub
LigD is thought to be recruited at DNA ends by the Ku protein which is bound to these DNA ends. Ku Bsub and LigD Bsub are expected to interact, as Ku Mtub and LigD Mtub , which were shown to interact in a yeast two-hybrid assay (28). To evaluate the effects of the different C-terminal truncations of Ku Bsub on the capacity to interact with LigD Bsub , we performed gel filtration experiments. Determination of elution volumes of Ku exCter and Ku core proteins and comparison with standard proteins allowed estimation of their molecular weight: 75 kDa and 68 kDa, respectively (SDS PAGE analyzes of the eluted fraction are presented in Figure 4 and chromatograms in Supplementary Figure S5). These estimations are in good agreement with the calculated molecular weight of Ku exCter and Ku core dimers, i.e. 61.2 kDa and 56.4 kDa, respectively. The LigD Bsub protein (calculated mass of 71 kDa) is eluted with an apparent molecular weight of 50 kDa, compatible with a monomeric status of the protein. In contrast, the estimated molecular weight of the dimeric Ku Bsub was ∼150 kDa which is more than 2-fold higher than the dimer calculated molecular weight (68 kDa). The elongated shape of Ku Bsub seen in SAXS experiment (see above) and the highly positively charged extended Cter domain could explain this apparent discrepancy.
Compared with LigD Bsub alone, the elution profile of the LigD Bsub mixed with Ku Bsub or Ku exCter proteins was more spread and was displaced toward the higher molecular masses (Figure 4 and Supplementary Figure S5), indicating that LigD Bsub interacts with Ku Bsub and Ku exCter. The elution volume of Ku Bsub or Ku exCter was not changed when mixed with LigD Bsub , suggesting that Ku Bsub (and Ku exCter) forms a more globular complex when interacting with LigD Bsub than the protein alone. By using labelfree surface plasmon resonance (SPR), the dissociation constants were determined by injecting a LigD Bsub solution on a sensor chip covered with one of the Ku proteins.  Figure S5). Using the SPR, in the same condition as the one used for Ku Bsub and Ku exCter, no dissociation constant could be determined (Supplementary Figure S6C). This result implies that the minimal Cter domain of Ku Bsub is required for interaction with LigD. This finding explains why Ku core mutant did not stimulate the end joining activity of LigD Bsub ( Figure 2D).

The extended C-terminal region and the core domain both contribute to Ku Bsub binding to dsDNA in absence of free ends
As the decreased stimulation of LigD Bsub by Ku exCter relative to Ku Bsub could result from an alteration of DNA binding, we compared the DNA binding properties of Ku Bsub , Ku exCter and Ku core proteins.
These proteins were mixed with supercoiled (sc) pZE14 plasmid and binding was analyzed by EMSA as described in Materials and Methods. Two types of retardation patterns were observed, a slightly shifted and discrete band migrating just above the free scDNA substrate and a considerably shifted smear of DNA ( Figure 5A). These two types of patterns were observed for Ku Bsub , Ku exCter and Ku core proteins, indicating a shared ability to bind scDNA. However, the concentration of Ku protein required to shift all DNA molecules was 0.5 M for Ku Bsub and 4 M for Ku exCter and Ku core, indicating that the C-terminally truncated mutants have a lower affinity for scDNA relative to the full size protein. The larger nucleoprotein complexes that migrated as smears in the gel formed with the three variants of Ku exhibited a similar difference in affinity. Re It has been proposed that the lysine rich extended Cter region of Ku Paer and Ku Msme could make electrostatic interactions with DNA (7,27). If this is true, adding the extended Cter to a protein without particular affinity for DNA should result in this protein gaining DNA binding ability. To assess this possibility, we fused the Ku Bsubextended Cter domain (KC) to the C-terminal domain of the Green Fluorescent Protein (GFP). The purified GFP-KC chimera and the wild-type GFP were incubated with the supercoiled pZE14 plasmid. In our conditions, this plasmid was weakly shifted by the GFP protein only at the most elevated concentration used (10 M, Figure 5F). Remarkably, a large fraction of the scDNA was shifted by 1 M of the GFP-KC protein (the lower concentration tested) and the DNA was entirely shifted with 3 M. Interestingly, retardation patterns obtained with supercoiled or linear DNA by different concentrations of GFP-KC were similar with all the DNA substrate shifted at 3 M of GFP-KC. This observation indicates that the presence of DNA ends does not change the affinity of the extended Cter region of Ku Bsub for dsDNA. Altogether, these results indicate that Ku Bsub binding to DNA without free ends is mediated by both the extended Cter and the Ku core domains.

Ku Bsub and C-terminally truncated Ku proteins thread inward linear DNA molecule after entering via the ends
We tested the ability of Ku Bsub and truncated mutants to bind linear dsDNA. A blunt-ended 5 -phosphorylated 279 bp DNA substrate (a DNA molecule without 5phosphorylated ends gave identical results, not shown) was incubated with each of the three Ku protein variants. Ku Bsub and both truncated mutants led to the formation of multiple discrete retarded DNA bands showing that all these proteins were able to bind linear dsDNA molecules (Figure 6A). The molecular weight of Ku-DNA complexes increased with increasing protein concentration. At 25 and 50 nM of proteins the number of retarded species was 3 and 4, respectively for all protein variants, suggesting that they display similar affinity for DNA ends. Increasing the concentration up to 400 nM yielded a single thick shifted band for Ku core and Ku exCter and nucleoprotein complexes that did not enter the gel for Ku Bsub . This result shows that DNA molecules with free ends can bind several C-terminally truncated Ku proteins until a maximum is reached. Proportions of unbound DNA were 60, 58 and 40% with 50 nM of Ku Bsub , Ku exCter and Ku core proteins, respectively. Interestingly, when the concentration of protein was doubled, the fraction of unbound DNA remained similar whereas the number of retarded species increased and the amount of DNA in the two less retarded bands clearly decreased on the gels. This observation suggests that the DNA binding of Ku Bsub and both truncated mutants at DNA ends is cooperative, as previously described for the human Ku (49).
To investigate the nature of these nucleoprotein complexes, we analyzed the binding of the three Ku protein variants on a linear dsDNA by TEM ( Figure 6,   (white arrows on Figure 6B1 and B2), demonstrating that Ku Bsub is loaded onto DNA ends, and (ii) Ku Bsub -DNA networks mediated by intra-and intermolecular bridging events based on protein-protein or/and DNA-protein interactions ( Figure 6B3, Supplementary Figure S7). These bridging events led to networks formation and concentration of DNA ends at the heart of these networks ( Figure  6C and Supplementary Figure S7). An increase of Ku Bsub concentration increased the size of these networks and decreased the number of Ku Bsub -DNA molecules not trapped into networks. At 300 nM, the networks led to the formation of aggregates ( Figure 6D). For both truncated Ku proteins, we did not observe protein-DNA networks but only DNA molecules covered by Ku mutants, predominantly at the ends, and naked DNA molecules ( Figure 6, panels E1-J). This suggests that the extended Cter domain mediates the assembly of Ku Bsub -DNA networks. Remarkably, the length of Ku exCter threads increased with protein concentration until the threads covered nearly all the DNA molecule (compare panels E1 with G). The Ku core protein displayed the same behavior as Ku exCter ( Figure 6, panels H-J). These results demonstrate that the C-terminally truncated Ku proteins are loaded onto DNA ends, as observed with full size Ku Bsub , and they reveal inward threading of Ku onto linear DNA molecules, which could not be easily observed with full size Ku Bsub due to the formation of Ku Bsub -DNA networks at elevated concentrations.
In light of these results, the multiband pattern observed in EMSA experiments ( Figure 6A) could be interpreted as progressive inward threading of Ku protein variants onto the DNA with Ku entering via the DNA ends. The single retarded band obtained with 400 nM of the C-terminally truncated proteins could correspond to DNA molecules completely covered. The absence of visible retarded bands at 400 nM of Ku Bsub could be due to the formation of high molecular weight nucleoprotein complexes or aggregates, as observed in Figure 6 (panels C and D).

Truncation of the extended C-terminal region alters Ku Bsub ability to bridge linear DNA molecules
The ability of Ku Bsub to interact and form Ku Bsub -DNA networks with supercoiled and linear DNA molecules prompted us to test whether Ku Bsub was able to bridge two DNA molecules, a potentially important property to maintain both ends of a DNA break in close proximity. Using a previously described bridging assay (46), we generated a blunt-ended 1001 bp linear DNA molecule 5 -biotinylated at one end and 5 -phosphorylated at the other end (1001BP in Figure 7) that was attached to streptavidin coated magnetic beads. Ku Bsub was then loaded on the DNA substrate and the ability of the Ku Bsub -1001BP complex to form a bridge with another DNA molecule (5 -phosphorylated blunt-ended 1876 bp DNA fragment) was assessed by analyzing the DNA recovered from the beads on agarose gel as described in Materials and Methods (see Figure 7 panel B). Non-specific binding of DNA on streptavidin-coated beads was measured using a DNA fragment carrying 5phosphorylated ends on both extremities (1001PP, Figure  7B). After removing unbound molecules, we also analysed the protein content of beads after the incubation of Ku Bsub with the 1001BP substrate ('M' fraction in Figure 7A) and after the incubation of the 1876 bp ('E' fraction in Figure  7A).
With Ku Bsub , a larger amount of the 1876 bp DNA fragment was retained on beads attached to 1001BP compared to 1001PP (Figure 7B), while a fraction of the protein was associated only with beads that were attached to the 1001BP substrate ( Figure 7A). This indicates that Ku Bsub can bridge DNA molecules. In contrast, the Ku exCter and Ku core mutant proteins did not exhibit any detectable bridging activity as similar amounts of the 1876 bp DNA fragment were retained on beads attached to 1001BP or 1001PP (Figure 7B), under conditions where both mutant proteins associated only with beads attached to 1001BP ( Figure 7A). These results are consistent with the TEM results and confirm that the extended Cter of Ku Bsub is required to maintain DNA extremities in close proximity.

The extended C-terminal region acts to limit threading and to concentrate Ku Bsub at DNA ends
The TEM analysis of micrographs obtained with 30 nM of Ku proteins on linear dsDNA suggested that the nucleofilaments formed by Ku exCter and Ku core were longer than those observed by Ku Bsub (compare panels B, E and H in Figure 6). However, the formation of Ku-DNA networks even at this Ku Bsub concentration did not permit to quantify this difference. We searched for reaction conditions limiting the formation of Ku Bsub -DNA networks while maintaining threading inward the DNA molecules via their ends.
The increase of ionic strength (from NaCl 50 mM to 200 mM) resulted in a significant decrease of Ku Bsub -DNA networks (Supplementary Figure S8). In this reaction condition and at a concentration of 150 nM of Ku protein variants, we monitored the formation of Ku Bsub -DNA networks and aggregates (such as those presented in panels B3, C and D of the Figure 6). Because the number of molecules contained in these high molecular weight complexes cannot be defined, their kinetic of formation was measured by counting the number of naked DNA molecules and nucleoprotein complexes not trapped into these networks over time (called untrapped DNA molecules, untrap. DNA in Figure 8D). We found that the frequency of untrapped DNA molecules with Ku Bsub decreased significantly (Mann-Whitney U test, P < 0.05) by up to 50% of the initial quantity after 60 min of incubation, whereas this frequency remained stable after incubation with Ku exCter and Ku core for the same time. For incubation times longer than 15 min, the frequency of untrapped DNA molecules was significantly lower with Ku Bsub than with Ku exCter and Ku core (Mann-Whitney U test, P < 0.05). Thus, the formation of high molecular weight nucleoprotein networks mediated by Ku Bsub depends on the concentrations of salt and protein as well as on the incubation time.
We classified untrapped DNA molecules into four types: (i) naked DNA, (ii) complexes with proteins at one or (iii) both DNA ends, and (iv) complexes with protein inside the DNA molecule with one, two or without covered ends (Figure 8E). After 2 min of incubation, the majority of DNA molecules (>83%) were bound by Ku Bsub , Ku exCter and Ku core, at one or mainly both ends. A small percentage (3%) of the DNA molecules displayed proteins bound inside (see white arrow in Figure 8A, termed diffusion events in Figure 8E). In at least two out of three cases, these diffusion events were concomitant with one or both DNA ends covered, suggesting that threads of Ku were broken during sample preparation and/or that part of the thread diffused inside the DNA molecule. Interestingly, we observed that the population of DNA molecules bound at one or two ends by Ku Bsub was stable for 30 min (82 ± 7%) and decreased. The observation that the frequency of untrapped DNA molecules with Ku Bsub also decreased over time suggests that the DNA molecules covered by Ku Bsub at one or both DNA ends are preferentially trapped into Ku Bsub -DNA networks.
We determined the fraction of DNA covered by the three Ku proteins in untrapped DNA molecules (ratio of the sum of DNA length covered by Ku Bsub or truncated proteins over the total length of DNA molecule at each incubation time). The distribution is shown on Figure 8F. The mean fractions of DNA covered were 20 ± 6; 37 ± 13; 38 ±13; 48 ±13% for Ku exCter and 18 ± 5; 34 ± 6; 34 ± 7; 45 ± 7% for Ku core, for 2, 15, 30 and 60 min of incubation time, respectively. Interestingly, these values were significantly lower with the Ku Bsub protein (Mann-Whitney U test, P < 0.05) and demonstrated a more limited threading (13 ± 7; 17 ± 7; 23 ± 7; 20 ± 8%). In addition, for incubation times longer than 15 min, more than 50% of bound DNA molecules displayed ends covered on a length ranging from 250 bp up to 570 bp for both truncated proteins, whereas this proportion fell to 13% for Ku Bsub with a covered length ranging from 250 bp to a maximum of 480 bp. These results indicate that the extended Cter domain limits inward DNA threading of Ku Bsub .
The properties to bind to DNA ends and to thread inward the DNA molecule should allow Ku Bsub and C-terminally truncated Ku proteins to protect DNA from degradation by an exonuclease, as previously described for Ku from different bacterial species, but also to protect against cleavage by an endonuclease. Moreover, the limited threading capacity of full length Ku Bsub relative to that of Ku exCter, as observed by electron microscopy, could lead to different DNA protection efficiencies. We probed the capacity of Ku Bsub and Ku exCter proteins to protect DNA molecules from degradation by the T5 exonuclease. The 5 -phosphorylated blunt-ended 1001 bp linear dsDNA substrate was preincubated with increasing amounts of Ku Bsub or Ku exCter proteins and then subjected to the T5 exonuclease. Analyses of the DNA degradation patterns showed that Ku Bsub and Ku exCter were equally efficient to inhibit the T5 exonuclease and protect the DNA ends ( Figure 9A). This 1001 bp DNA molecule harbors two identical HincII sites, one positioned at 266 bp (hereafter named site 266) from one end and the other (named site 421) at 421 bp from the same DNA end ( Figure 9B). DNA substrate pre-bound with Ku Bsub or Ku exCter was digested by HincII and analyzed on an agarose gel ( Figure 9C). In the control reaction mixture where Ku proteins were omitted, the three main DNA bands expected from complete digestion of the two HincII sites were obtained (lane 2 in Figure 9C). Prebinding of Ku Bsub or Ku exCter on the DNA molecules led to several results. Firstly, Ku Bsub and the truncated mutant protein protected the DNA from HincII cleavage, as indicated by the absence of digested DNA molecules at elevated protein concentrations ( Figure 9C, lanes 8, 13, 14). Secondly, the concentration of Ku exCter required to protect 50% of the linear DNA substrate is approximately two times lower compared to Ku Bsub (around 1 M versus 2 M, see quantification in Supplementary Figure S9B). Thirdly, quantification of DNA amounts in bands corresponding to the 580 bp and the 266 bp HincII digested products provided a good estimation of the number of cutting events at site 421 and at site 266, respectively ( Figure 9D). The number of cutting events decreased more rapidly at the site 266 than at the site 421 when raising the concentration of Ku Bsub as well as of Ku exCter (compare dashed lines to full lines in Figure 9D). This result suggests that the site 266, the site closest from the end, was preferentially protected by Ku Bsub (and, to a lesser extent, by Ku exCter mutant), most probably by its threading from this end. Although the site 266 seemed to be protected with the same efficiency by Ku Bsub and the C-terminal mutant, this is not the case for the site 421. Indeed, lower concentrations of Ku exCter are  required to protect this site compared to Ku Bsub (compare grey full line to black full line in Figure 9D). In keeping with the electron microscopy experiments, this difference is consistent with the more limited threading capacity of Ku Bsub relative to the Ku exCter mutant.
The kinetic of DNA protection against HincII cleavage was determined under conditions where Ku Bsub and Ku exCter concentrations prevent total digestion after 2 h of incubation. Reaction samples were extracted at different times and analyzed to determine the fraction of uncut 1001 bp DNA molecule. In our testing conditions, HincII digested nearly all the 1001 bp DNA molecules in 30 min (Figure 9E). When 0.5 M of Ku Bsub was pre-incubated with the DNA, two phases was observed during the digestion kinetic. The first one is a rapid digestion of the DNA substrate in the first 30 min after addition of the HincII enzyme, leading to 34% of uncut DNA. Beyond 30 min, a very slow digestion phase was observed, leading to 25% of undigested DNA after 120 min. The same biphasic digestion kinetic was observed using 1.18 M of Ku Bsub except that the proportions of uncut DNA were 57% and 51% after 30 and 120 min, respectively ( Figure 9E). While 1.18 M of Ku exCter totally protected the DNA, a quite stable DNA protection was also measured with 0.5 M of Ku exCter beyond 30 min, after a first phase of rapid digestion leading to 68% of uncut DNA ( Figure 9E). Based on the observations presented above, these results strongly suggest that during the first phase of the kinetic, DNA molecules displaying HincII sites uncovered by nucleoprotein filaments formed after the pre-incubation with Ku Bsub (or Ku exCter) were rapidly cut by the endonuclease while those bearing HincII sites covered by Ku Bsub (or Ku exCter) remained mostly protected over the time. Then, the proportion of uncut DNA molecules observed during the second phase should reflect the proportion of DNA molecules with HincII sites protected by nucleoprotein filaments formed during preincubation with Ku Bsub or Ku exCter. Consequently, nucleoprotein filaments formed by Ku Bsub and Ku exCter appeared equally stable. Moreover, the higher amount of DNA molecules fully protected by 0.5 M of Ku exCter compared to 0.5 M of Ku Bsub indicates that nucleoprotein filaments formed by Ku exCter were longer than those formed by Ku Bsub at the same concentration, reinforcing results obtained by TEM experiments ( Figure 8F).
Ku Bsub and Ku exCter were also able to protect supercoiled dsDNA (not shown) and nicked circular dsDNA against cleavage by HincII at elevated concentrations (Supplementary Figure S9 panel A). However, for both proteins protection of the 1001 bp linear DNA molecule was more efficient than protection of the nicked circular molecule (Supplementary Figure S9 panel B). Moreover, the concentrations of Ku exCter and Ku Bsub needed to protect 50% of the circular DNA are quite similar (∼3 M and 3.5 M, respectively, Supplementary Figure S9B). These results support the notion that the higher protection of linear DNA by Ku exCter relative to Ku Bsub is due to a more efficient threading from DNA ends. Protection of circular DNA molecules by Ku Bsub and Ku exCter could be related to the formation of large nucleoprotein complexes at elevated concentrations (see Figure 5A) which could block accessibility of HincII sites.

DISCUSSION
While the lysine rich extended C-terminal regions of Ku Paer and Ku Msme are involved in the interaction of Ku with DNA, the role of this interaction in the NHEJ pathway remains unclear (7,27). In this study, we report that the Cterminal domain of Ku Bsub , which was delineated by an in silico analysis of prokaryotic Ku proteins, is composed of two regions (Figure 1) with distinct functions. The minimal C-terminal domain following the Ku core domain is required for interaction with LigD Bsub (Figure 4) and for the Ku-dependent stimulation of the ligase activity ( Figure 2). Next, the lysine rich extended C-terminal region increases the Ku-dependent stimulation of LigD Bsub (Figure 2). Characterization of the DNA binding properties of Ku Bsub truncated mutants shows that the extended Cter region is required for the formation of Ku Bsub -DNA networks with linear as well as circular dsDNA (Figures 5 and 6) and for the bridging of DNA molecules (Figure 7). Based on the SAXS analysis and molecular modeling of the dimeric Ku Bsub , showing a ring shape with extended C-terminal arms, electron microscopy analyses of the Ku Bsub -DNA interaction and DNA protection against endonuclease cleavage (Figures 3, 6 and 9), we propose that Ku Bsub binds dsDNA ends and threads inward the DNA molecule, as its eukaryotic homologue. Finally, we demonstrate that the basic extended Cter domain acts to limit threading of Ku Bsub inward the dsDNA (Figures 8 and 9).
Taken together, these results lead us to propose several roles for the Ku C-terminal domain of B. subtilis. Firstly, its affinity for linear as well as circular DNA allows the recruitment of Ku Bsub to unbroken DNA, placing Ku Bsub in a suitable position for efficiently handling DNA ends in the cell, as already suggested for the Ku Msme protein (27). When a DSB occurs, Ku binds the broken ends and threads inward the chromosome, a process requiring the addition of new Ku Bsub molecules at the DNA ends. Secondly, the extended Cter domain limits the extent of threading probably by interacting with the DNA onto which Ku Bsub is loaded (see Supplementary Figure S7 panel E; other explanations for inhibition of Ku threading are discussed below). Finally, LigD Bsub will be recruited at the DSB site by the minimal Cter region of Ku Bsub proteins bound near DNA ends. Thus, by limiting threading, the extended Cter domain favors accumulation of Ku Bsub within a short distance of the DSB and the recruitment of LigD Bsub at this site for repair.
Based on our data, other roles could be proposed for the extended Cter domain of Ku Bsub . The formation of Ku Bsub -DNA networks (see electron micrographs Figure 6 panels B3 and C) suggests that the extended Cter could also play an active role in bringing DNA molecules together, potentially maintaining DSBs in close proximity to facilitate sealing by LigD Bsub . This hypothesis is reinforced by the requirement of the extended Cter to bridge two dsDNA molecules in absence of LigD Bsub (see Figure 7). However, the fact that Ku exCter is only slightly altered in its ability to stimulate the ligase activity of LigD Bsub compared to Ku Bsub ( Figure  2) suggests that the ability to bridge two DNA molecules together is not absolutely required to perform an efficient ligation, at least in our in vitro conditions. However, we cannot rule out that in germinating spores where NHEJ is active (50), the ability of Ku Bsub to bridge DNA molecules could stimulate the ligase activity of LigD.
Based on the DNA bridging assay, Ku Bsub could bridge two linear DNA molecules by their ends (end-to-end bridging) and/or by their internal parts (adjacent bridging). By TEM, we did not observe end-to-end bridges (demonstrated, for instance, by the visualization of DNA molecules bridged by their ends with a Ku nucleofilament located at the junction). However, we have visualized molecules interacting by their end-proximal internal parts covered by Ku Bsub (Supplementary Figure S7 panels A1, B and E). These observations favor an adjacent bridging mechanism dependent of the Ku Bsub extended Cter domain which could be a primary step toward an end-to-end repositioning required for an efficient ligation. These DNA bridging sequential events have been proposed for the human NHEJ machinery in vivo (51). Finally, the human Ku heterodimer binds DNA with a defined polarity, Ku70 and Ku80 being located respectively proximal and distal to the DNA end (21,52). It is currently not known whether the bacterial Ku protein binds DNA ends in a preferred orientation. However, if the C-terminal domain played a crucial role in determining binding polarity, it is likely that the Ku Bsub Cterminal domain mutants would have been affected in their threading abilities. The similar stability of the nucleoprotein filaments formed by Ku Bsub and the C-terminal mutants as well as their conserved ability to thread is not in favor of such a role for the C-terminal domain of Ku Bsub .
The ubiquitous presence of the minimal Cter region in the prokaryotic Ku proteins (Figure 1) strongly suggests that its role in the Ku Bsub -LigD Bsub interaction is evolutionary conserved. The slight increase of the stimulation of LigD Paer using a Ku Paer mutant truncated of its extended Cter region compared to a mutant similar to the Ku core protein used here (7) shows that the minimal Cter of Ku Paer is also involved in the stimulation of LigD Paer . The fact that this domain is important for the LigD Bsub and LigD Paer stimulation ( Figure 2 and (7)) as well as for the formation of the Ku Bsub -LigD Bsub complex ( Figure 4) prompt us to propose that its role in promoting the Ku-LigD interaction is conserved in prokaryotes. The segment encompassing residues 111 to 273 of Ku Mtub was sufficient to interact with LigD Mtub in a yeast two-hybrid assay (28). This segment contains the minimal Cter region (see Figure 1) which is in agreement with our hypothesis.
The prevalent addition of the basic extended tail to the C-terminus of bacterial Ku proteins suggests also that its role is conserved. This lysine-rich C-terminal sub-region is involved in the interaction of Ku Paer , Ku Msme and Ku Bsub with DNA and its basic property suggests that it interacts with this molecule (7,27). We demonstrate this hypothesis by showing that addition of the extended Cter of Ku Bsub to the GFP protein allows the resulting GFP-KC chimera to interact with DNA ( Figure 5). Moreover, we show by electron microscopy that this extended domain limits the DNA threading ability of Ku Bsub (Figure 8). Interestingly, Ku Mtub does not contain a basic extended Cter region and it was proposed to translocate along linear dsDNA molecule (6). The ability of Ku Msme for inward translocation from DNA ends remains to be investigated but a Ku Msme truncated for its lysine rich C-terminal domain is able to translocate along a linear DNA molecule (27). Finally, the threading ability of Ku Paer was not visualized by EMSA experiment using a 2.7 kbp linear dsDNA (7). However, using the same methodology, we were able to observe multiple discrete retarded bands (corresponding certainly to the threading of Ku Bsub inward the DNA molecules as demonstrated by TEM in Figure 6) only with linear dsDNA substrate lesser than 500 bp length (not shown). Then, most of the bacterial Ku proteins, if not all, are able to thread inward a linear dsDNA molecule as their eukaryotic Ku70/80 homologues and we propose that the addition of a basic extended Cter domain limits (or prevents) this translocation mechanism. While the human Ku protein has been shown to bridge two DNA molecules in vitro (53), we discovered that a bacterial Ku protein also exhibited this activity. Future studies will be needed to determine whether the bridging activity is conserved across bacterial Ku proteins.
The threading ability of eukaryotic Ku proteins is proposed to increase the efficiency of the NHEJ mechanism by allowing the binding of other NHEJ factors at the DNA ends (25) and by displacing other proteins bound to the DNA such as histones (26). Similar roles can be hypothesized for the threading of Ku Bsub on DNA, i.e. allowing the binding of LigD Bsub and its accessibility to DNA ends and displacement of other proteins bound in the vicinity of the DSB. Since bacterial Ku (at least Ku Bsub and Ku Paer ) display a 5 -dRP lyase activity on abasic site (17) as the human Ku70/80 (16), translocation inside the DNA molecule could also increase the repair of abasic site surrounding a DSB. Finally, dsDNA protection against exonuclease and endonuclease activities in vitro by Ku Bsub (Figure 9) suggests that Ku could protect the DNA ends of a broken chromosome against degradation by cellular enzymes. So, why did B. subtilis (and probably most of the NHEJ capable bacteria) conserve a domain limiting this threading ability?
Although no data supports a putative toxic effect of threading, one can imagine that translocation of Ku inside a broken chromosome could affect the genome integrity by displacing useful DNA repair proteins or by creating roadblocks against other cellular machineries. Thus mechanisms to regulate threading should exist. Eukaryotic Ku70s harbor a SAP domain at their C-terminal ends. This domain is thought to interact with the DNA (54) and could be important for the control of the threading ability of the Ku heterodimer (21). This view is consistent with structural changes of this extension observed upon DNA binding in the human Ku heterodimer, suggesting a direct interaction between the DNA molecule and this SAP domain (55,56). Also, the binding regulation of the yeast Ku to DNA could involve the sumoylation of the Ku70 Cterminal domain (57). However, the absence of a Ku mutant with an altered translocating ability does not allow demonstrating this threading regulation hypothesis. Besides this putative regulating SAP domain, it has been shown that the threading ability of the human Ku70/80 inside the DNA molecule is finely controlled by the DNA-PK catalytic subunit (58), strongly suggesting that ways to regulate the threading mechanism are required to allow an efficient NHEJ process and/or preventing toxic effect in eukaryotes. Prokaryotic Kus, with the exception of one Ku from Streptomyces coelicolor, do not contain the SAP domain (19,20) Nucleic Acids Research, 2016, Vol. 44, No. 10 4805 and no prokaryotic homologue of DNA-PKcs has been identified. Then, we propose that the extended Cter region of Ku Bsub , and possibly of other bacterial Ku, has evolved to keep a Ku-threading regulating function, as it is probably the case for the eukaryotic SAP domain. The molecular mechanism by which this regulation occurs could be electrostatic interactions between this lysine rich tail and the phosphodiester backbone of a DNA molecule, as suggested for Ku Paer (7). Such interaction between this domain and the encircled DNA molecule should limit the threading of Ku inward the DNA, a phenomenon amplified by an increase of the length of the thread. We cannot exclude that differences in length of nucleoprotein filaments formed by Ku Bsub and the C-terminal deletion mutants could be due to altered on-and/or off-rate constants of binding to DNA ends in the mutants. However, two lines of evidence do not support such a hypothesis: (i) the number of retarded bands observed by EMSA with the different Ku proteins were similar at concentration allowing a limited number of binding events (3 at 25 nM of proteins, Figure 6A) and (ii) a similar stability of the filaments formed by these Ku proteins that protect DNA from endonuclease cleavage was observed ( Figure 9D).
Finally, this Ku-threading regulating role of the extended Cter as well as its role in the bridging of DNA molecules raise the question of the efficiency of the NHEJ pathway in bacteria encoding a Ku protein without this region, such as the Ku from M. tuberculosis, as well as the way to control the bridging and threading abilities of Ku in these bacteria.