FIG. 4.

Ligation time course of S. aureus DNA ligase (0.25 μM) with 17 μM ³²P-labeled nicked substrate (see Materials and Methods). Activity is measured in relative densitometric units of ligated product (59-mer) on a 10% acrylamide, 8 M urea gel. (A) Lanes 1 to 7 represent 0, 2, 4, 8, 18, 40, and 60 min of incubation at 30°C. (B) Gel assay comparing activity of full-length S. aureus DNA ligase with the purified N-terminal and C-terminal domains over 60 min at 30°C. Lane 1, no enzyme control; lane 2, 0.25 μM full-length ligase; lane 3, 0.25 μM 40-kDa N-terminal domain; lane 4, 0.25 μM 30-kDa C-terminal domain; and lane 5, 0.25 μM concentration of each of the N- and C-terminal domains, respectively. Lanes 6 to 8 represent the N-terminal, C-terminal, and both the N- and C-terminal domains in excess at 0.25 mM, respectively.