The competitive interaction between HosA and T4 DNA ligase with the nicked TFBS of HosA was examined using d-PAGE. TL, TR, and TH with a ratio of 1:1:1 were annealed to form the DNA template. Different concentrations of HosA (0, 0.168, 0.335, 1.68, 3.35, 16.75, and 33.5 μM) and 200 nM DNA template were then incubated in 20 μl of 1× T4 DNA ligase buffer at 25°C for 20 min to allow complete interaction between protein and DNA. After that, 4 U of T4 DNA ligase was added to the DNA-HosA complex with an incubation period of 30 min at 37°C. Then, the reaction was terminated by heating at 85°C for 10 min and was immediately cooled on the ice. The complexes were then mixed with 1× d-PAGE loading buffer. For d-PAGE assay, the samples were loaded on a 15% d-PAGE gel [volume (5 ml): 15% acrylamide (acrylamide and bis-acrylamide at a ratio of 29:1), 5× TBE (1 ml), tetramethylethylenediamine (TEMED; 2.5 μl), and 10% ammonium persulfate (25 μl), and 7 M urea]. The electrophoresis was run at a constant current of 5 mA for about 20 min, using 1× TBE as running buffer. The gels were stained with SYBR Gold for 40 min and then photographed under a Transilluminator.

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