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).