Figure 2.

p18CycE inhibits in vitro DNA ligation and plasmid reactivation. (A) Nuclear cellular extracts from HEK 293T transfected with increasing amounts of HA p18CycE (lanes 1–3) and HA CycE (lanes 4–6) were incubated with a pUC18 plasmid DNA linearized with the EcoRI restriction enzyme. The reaction was incubated at 37°C to allow the DNA to be re-ligated. T4 DNA ligase reaction and linearized pUC18 were used as controls (lane 7–8). The DNA ligation reaction was separated by gel electrophoresis. Expression of the transfected constructs was determined by immunoblotting with anti-HA antibody (right panel). (B) Nuclear cellular extracts from HEK 293T cells transfected with EGFP or EGFP p18CycE, or untransfected (UT) were used as described earlier in the text (A) for in vitro DNA ligation. Lanes 2 and 3 (left panel) represent identical reactions. The right panel shows the expression of EGFP and EGFP p18CycE immunoblotted with anti-EGFP antibody. β-actin was used as a loading control. (C) The DNA ligation reaction mix was transformed into E. coli that were then plated on agar plates containing Amp. The number of colonies was expressed relative to the percentage of those obtained with the T4 DNA ligase-treated DNA that were considered to be 100%.