Fig. 4: Nucleic acid and cofactor-specific binding to DNA ligase measured using the SDR assay.
From: A general assay platform to study protein pharmacology using ligand-dependent structural dynamics
a DNA ligase crystal structures illustrating the relative positions of the N- and C-termini, DNA and nucleotide binding sites for the E. coli ligase (MW 75 kDa) complexed with NAD+ (PDB 5TT5) from Unciuleac MC et al., with NAD+ and DNA (PDB 2OWO) from Nandakumar J et al., and bacteriophage T7 ligase (MW 41.1 kDa) complexed with ATP (1A0I) from Subramanya HS et al. The structure of NAD+ is shown. b Agarose gel electrophoresis of HindIII-digested λDNA repair by ligases from E. coli and bacteriophage T7 containing N- or C-terminal HiBiT α-peptide. Ligase concentrations used: 1, NEB E. coli ligase, 0.5 U/μL; 2, E. coli N-HiBiT, 1 µM; 3, E. coli C-HiBiT, 100 nM; 4, NEB T7 ligase, 150 U/ μL; 5, T7 N-HiBiT, 100 nM; 6, T7 C-HiBiT, 1 μM. Control ligases were from NEB and used as directed. Gel is representative of 2 replicates. Concentration-response curves obtained for the SDR assay for 0.5 nM E. coli Lig-N-HiBiT (solid circle), 1 nM E. coli Lig-C-HiBiT (open circle) and 1 nM T7 Lig-N/C-HiBiT (solid/open squares, respectively). c, d a 22-mer dsDNA oligo; e, f NAD+; g, h ATP. Error bars are the SD, n = 3 experiments. Supplementary Table 1 supports panels (c−h). kb, kilobase; Lig, ligase. The uncropped gel and source data are provided in a Source Data file.
