Figure 3.

Replication of the plasmid DNA assembled with pre-RCs in NPE. (A) DNA replication products analyzed by agarose gel electrophoresis. Conventional pre-RC and GAL4 pre-RC were assembled by incubating the bead-coupled plasmids (3.0 kbp) at 23°C for 30 min in mock-depleted and Cdc6-depleted HSS, respectively. We then added 2 vol of NPE containing [α³²P]-dATP to 1 vol of the assembly mixture, and the reaction was terminated at the indicated time by isolating DNA. The purified DNA was separated by agarose gel electrophoresis, and the replication products were visualized by autoradiography. The plasmid without the GAL4-binding sites was used as a negative control (-preRC) and 500 μM roscovitine was added to inhibit the initiation of DNA replication (+). (B) Graphic presentation of quantified data from (A). Total amounts of [α³²P]-dATP incorporated into DNA were measured and plotted against incubation time in NPE.