Oligomerisation of Ku from Mycobacterium tuberculosis promotes DNA synapsis

Abstract

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is estimated to infect nearly one-quarter of the global population. A key factor in its resilience and persistence is its robust DNA repair capacity. Non-homologous end joining (NHEJ) is the primary pathway for repairing DNA double-strand breaks (DSBs) in many organisms, including Mtb, where it is mediated by the Ku protein and the multifunctional LigD enzyme. In this study, we demonstrate that Ku is essential for mycobacterial survival under DNA-damaging conditions. Using cryogenic electron microscopy (cryo-EM), we solved high-resolution structures of both the apo and DNA-bound forms of the Ku-Mtb homodimer. Our structural and biophysical analyses reveal that Ku forms an extended proteo-filament upon binding DNA. We identify critical residues involved in filament formation and DNA synapsis and show that their mutation severely impairs bacterial viability. Furthermore, we propose a model in which the C-terminus of Ku regulates DNA binding and loading and facilitates subsequent recruitment of LigD. These findings provide unique insights into bacterial DNA repair and guide future therapeutics.

Fig. 1. The importance of Ku for mycobacterial survival under DNA double-strand break conditions and cryo-EM of Apo-Ku-Mtb.

M. smegmatis strains from stationary phase were exposed to 0.1% (v/v) MMS (A) or subjected to desiccation for 24 h. B CFU counts were determined before and after treatments on 7H10 agar. Δku – ku deletion mutant, ΔkupMV306 - ku deletion mutant with empty plasmid; ΔkupMV306::kusm – complemented ku deletion mutant containing pMV::msmeg5580. Results of two independent experiments, done with three biological replicates. ns – non-significant, **** p < 0.0001, one way ANOVA or unpaired t test. C Representative 2D class averages of apo-Ku-Mtb. D Cryo-EM map and model for apo-Ku-Mtb. The map is shown as a transparent grey to 4.04 Å resolution. Protomer A is shown in pink and protomer B in blue, the C-terminal extensions are shown in darker colours. E) Domain organisation schematic for Ku-Mtb and human Ku70/80. Ku-Mtb is shown in pink and blue. Ku70 vWA is shown in yellow and the DNA binding domain in orange. Ku80 vWA is coloured in lime green and the DNA binding domain in dark green. F) A comparison of Ku-Mtb (This study) and human Ku70/80 (PDB:1JEY). The models are coloured according to E.

Fig. 2. Ku-Mtb binding to DNA.

A EMSA gel of DNA with increasing ratio of Ku protein. B Representative cryo-EM micrograph showing filaments of Ku-Mtb. C Representative 2D class averages. D Cryo-EM map and model of Ku-Mtb with DNA showing the repeating unit of two Ku homodimers bound to DNA. Protomer A in pink, protomer B in blue and DNA in grey and map at 2.96 Å resolution coloured according to chain colour. Insets show interfaces between protomers with one binding to metal ion. E Two views of an extended model of the Ku-Mtb filament, showing ~40 Å distance between DNA ends.

Fig. 3. Ku-Mtb binding DNA.

A Mass photometry of Ku binding to DNA, with increasing ratios of Ku:DNA and increasing molecular weight peaks observed with models above each shown. B FIDA experiment, showing the hydrodynamic radius (Rh) (nm) as Ku protein concentration is increased with DNA. No fits are added due to it not being a 1:1 binding event. Plateaus in the Rh value are shown how they correspond to the different cryo-EM structures observed.

Fig. 4. Disruption of Ku oligomerisation.

A Apo-Ku-Mtb model with protomer A coloured pink, with the C-terminal linker and a-helix coloured purple, protomer B is coloured in blue with the C-terminal linker and a-helix coloured dark blue. Inset, shows the interface between the C-terminal a-helix and Ku which blocks filament formation in the apo structure. Residues are shown as sticks and labelled. B Ku-Mtb DNA filament model showing interfaces between Ku-Ku molecules. The central Ku homodimer is coloured pink for protomer A and blue for protomer B. The two Ku molecules either side are coloured light pink for protomer A and light green for protomer B. The DNA is coloured grey. Inset left, shows a close-up view of the Ku interface at the gap between the DNA ends, with residues shown as sticks and labelled. Inset right, shows a close-up view of the Ku interface between the DNA engaged interface with residues shown as sticks and labelled. C Representative cryo-EM micrograph for the L13A/V14A Ku-Mtb double mutant. D Representative 2D class averages for the L13A/V14A Ku-Mtb double mutant. E Cryo-EM map and model for the L13A/V14A Ku-Mtb double mutant to 3.67 Å resolution. Protomer A is coloured pink and protomer B blue, with the DNA shown in grey. F Survival percentages of M. smegmatis strains exposed to 0.1% (v/v) MMS for 6 h or G) desiccated for 24 h. Δku – ku deletion mutant, ΔkupMV306 - ku deletion mutant with empty plasmid; ΔkupMV306::kusm – complemented ku deletion mutant containing pMV::msmeg5580, ΔkupMV306::kusm LV – ku deletion mutant containing pMV::msmeg5580 with L23A/V24A mutations. Percentage survival was calculated by dividing CFU ml⁻¹ after treatment by CFU ml⁻¹ before treatment, multiplied by 100. Results of three independent experiments, performed with three biological replicates, error bars show SEM. ns – non-significant, *** p < 0.001, ** p,0.01, unpaired t-test, unpaired t test; ns – non-significant (p > 0.05, one way ANOVA or unpaired t test).

Fig. 5. Positive stain of Ku-Mtb.

Electron micrographs of DNA-KuMtb complexes obtained at 200 nM Ku with blunt 401 bp (a-e) and 1440 bp (f) fragments. The samples were analyzed in positive staining and darkfield imaging mode. (a) 440 bp control DNA fragments (b) Ku filaments at one end (b1) or at two ends (b2-3); The arrows indicate Ku filaments. (c 1–4) circularization of DNA fragment mediated by end-to -end joining. (d1-4) circularization of DNA fragment mediated by bridging B1 where the two filaments are in the same orientation. (e 1–4) circularization of DNA fragment mediated by bridging B2 where the two filaments are in opposite orientation. (f1) 1440 pb DNA fragment linked by Ku at one end and (f2) circularization of DNA fragments mediated by Ku. The two scale bars correspond to 50 nm.

Fig. 6. Model of the NHEJ mechanism in Mtb.

Apo-Ku-Mtb is first shown in grey with the C-terminal helices coloured in blue and pink. Ku-Mtb then binds to DNA loading and forming an oligomer, with the C-terminal helices then released to allow filament formation. LigD is then finally recruited and DNA ligated in an unknown mechanism. An AlphaFold 3 model of LigD is shown and coloured according to three domains, ligase, PE and polymerase.