Figure 7.

EMSA of Tth DNA ligase bound to duplex DNA. (A) Effects of minor groove interactions as well as Mg²⁺ on Tth ligase binding and ligation. DNA samples (100 fmol) were incubated with 100 fmol of Tth DNA ligase at 26.5°C in the presence or absence of Mg²⁺ in 20 μl total volume. After 5 min incubation, half of the samples were adjusted to 10% glycerol, and electrophoresed through a 10% native gel to detect ligase binding. The other half were heat-denatured, and resolved on a 20% denaturing gel to detect ligation. (B) Tth DNA ligase binds to duplexes containing the uracil:purine base pair on the 3′-hydroxyl end. The experiments were performed with equimolar DNA substrates and the ligase (100 fmol each) at 26.5°C for 5 min in the absence of Mg²⁺. 3′ (5′)-X:Y indicates that X is on the 3′-hydroxyl (5′-phosphate) end of the ligase junction, and Y is on the template.