Fig. 3

ComH is a double-stranded DNA- binding protein. a Visualisation by electrophoretic mobility shift assays of nucleoprotein complexes formed by ComH-His₆. Increasing concentrations of purified ComH-His₆ (0, 0.5, 1, 1.5, and 2 µM) were incubated with Cy5-labelled dsDNA (18 bp) and ssDNA (18-mer) substrates (30 nM). The free DNA substrates and nucleoprotein complexes were resolved by native PAGE (6%). b Comparison of ComH-His₆ binding to dsDNA (50 bp) and ssDNA (50-mer) by Surface Plasmon Resonance. Increasing concentrations of ComH-His₆ (0, 0.31, 0.62, 1.25, 2.5, and 5 µM) in binding buffer (PBS + 0.005% Tween20) were injected onto the streptavidin chip containing immobilised DNA substrates. The SPR derived sensograms for dsDNA and ssDNA are shown. c Interaction measurements by microscale thermophoresis of the affinity between ComH-His₆ and a 18 bp DNA labelled with a FAM in 5’. All experiments were performed in duplicate and error bars = SD. d Analyses by sedimentation velocity analytical ultracentrifugation (SV-AUC) of the interaction between ComH and 18 bp dsDNA-FAM. The free dsDNA-FAM has a sedimentation coefficient of 2.1S. ComH forms a main complex with a sedimentation coefficient of 6.5S.