Journal: bioRxiv
Article Title: It takes two (Ku) to repair: Mechanistic insights into the minimal NHEJ system of Mycobacterium Tuberculosis
doi: 10.64898/2025.12.16.694593
Figure Lengend Snippet: Mutants of Mtb LigD interact with Mtb Ku, and show distinctive ligation activities. a) Predicted interactions between the Ku C-terminal region and LigD domains from M. tuberculosis . AlphaFold2 (AF2) models of LigD and its domains. 1) Domain organization of LigD, including the polymerase domain (PolDom, blue), ligase domain (Lig, yellow) containing an OB subdomain (red), and phosphoesterase domain (PE, green). 2) Predicted interaction between the Ku C-terminal region (residues 243-273, salmon) and the LigD PolDom (residues 1-290, blue). Residues at the interface are highlighted and labeled, with interface confidence scores of AF2 ipTM = 0.87 and actfipTM = 0.86. 3) Predicted interaction between the same Ku C-terminal region and the LigD Lig domain (residues 445-640, yellow), showing key contact residues. Interface confidence scores are AF2 ipTM = 0.79 and actfipTM = 0.77. Insets in Middle and Right panels show detailed views of the Ku C-terminal helix (salmon) docking onto each LigD domain. The amino acids that were mutated are represented as large spheres and in a different color (cyan and orange for PolDom and Lig domains, respectively). b) Mtb LigD WT and mutants interact with Mtb Ku. The 6 Mtb LigD mutants (polymerase domain: D162R, V194D, R198E and ligase domain: D522R, K579E and L580E) were pulled down with anti-SNAP beads. Proteins were identified on a denaturing SDS-PAGE followed by a silver stain. Mtb LigD MW= 81kDa, SNAP- Mtb Ku MW= 53kDa, SNAP peptide MW= 20kDa. c) Mtb LigD mutants show a wide variety in ligation efficiency. Mtb Ku and 1kb DNA (NoLimits) were incubated at a two homodimeric Mtb Ku/DNA-end ratios followed by the addition of one Mtb LigD/DNA-end. Reactions were deproteinated and ligation products were analyzed on a SYBR Gold post-stained 0.8% agarose gel.
Article Snippet: For each reaction, 3 μL SNAP-Capture Magnetic Beads (New England Biolabs, #S9145S) were washed twice with 100 μL of Buffer L and resuspended in 100 μL of Buffer L. 10 μM SNAP-Ku or SNAP peptide (New England Biolabs, # P9312S) were added to the beads for immobilization and incubated in a thermo-shaker overnight at 4°C, 600 RPM.
Techniques: Ligation, Labeling, SDS Page, Silver Staining, Incubation, Staining, Agarose Gel Electrophoresis
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