FIG. 7.

NAD⁺-dependent DNA ligase activity of Lig_Tk. (A) Ligation reactions were carried out at 80°C with 0.1 mM ATP, with 0.1 mM NAD⁺, without cofactor, or with 0.1 mM ADP for 0, 15, and 120 min. (B) Ligation reaction measurements using hexokinase-treated NAD⁺. All ligation reactions were carried out at 80°C for 2 h. Lane 1, 0.1 mM ATP without hexokinase treatment was added to the ligation reaction mixture; lane 2, 0.1 mM ATP treated with hexokinase for 2 h was added to the ligation reaction mixture; lanes 3 and 4, 0.1 mM NAD⁺ treated with hexokinase for 2 h was added to the ligation reaction mixture. The mixture was applied to the gel prior to the ligation reaction (lane 3) and after the ligation reaction (lane 4).