Figure 5.

Unwinding of a site-specific origin upon activation of GAL4 pre-RC. (A) Schematic of P1 nuclease assay for unwinding of plasmid DNA after activation of pre-RCs in NPE. P1-treated DNA was further digested with BamHI or MfeI that recognizes different unique digestion sites of the plasmid. BamHI and MfeI sites are located at the proximal side (at the edge; 0 kb distance) and opposite side (3.0 or 3.3 kb distance) of the DNA-binding sites, respectively. (B) The bead-coupled plasmids assembled with conventional (C) or GAL4 pre-RC (G) were incubated in NPE containing 67 ng/μl aphidicolin at 23°C for 30 min to allow unwinding of dsDNA. Plasmids bound with GAL4-GST were used as a negative control of pre-RC assembly (N). After incubation, the plasmids were isolated and treated with P1 nuclease to digest ssDNA regions of the plasmids. P1-treated DNA was then purified and further digested with BamHI or MfeI. The purified DNA from the restriction enzyme digestions was subjected to agarose gel electrophoresis and visualized. (C) The distribution of DNA fragments obtained after P1 nuclease followed by the restriction enzyme digestions from a single experiment. The amount of DNA was estimated from the staining of DNA in agarose gel with the aid of the Image J program and shown as brightness. The peak values of each sample were taken as 100%. Reproducibility of the data is shown in Figure S5.