FIGURE 5.

DNA end joining activity is stimulated by the DNA-binding domain of LigIII. A and B, to measure the stimulation of CC ligation activity by DBD and ZnF-DBD, purified CC (100 nm) was mixed with nicked, radiolabeled DNA (4 nm) and increasing concentrations (1, 2.5, 5, 7.5, 10, 20, 30, 40, 60, and 80 μm) of the DBD (A) or ZnF-DBD (B). The extent of ligation at various times (30 min, 1, 2, 3, 6, or 9 h) was determined by periodically removing and quenching aliquots of the reaction and measuring the extent of conversion of a 15-mer substrate to 28-mer ligated product. C, ligation activity of the CC is stimulated when the DBD is added in trans (open circles) but inhibited when the ZnF-DBD is added in trans (closed circles). D-F, DNA binding by the CC (D), ZnF-DBD (E), and DBD (F) was measured using fluorescence anisotropy. The affinity for nicked DNA (•), intact DNA (○), nicked DNA lacking a 3′hydroxyl (▾), and nicked DNA lacking a 5′ phosphate (▵) was measured.