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. 2015 Jan 20;14(7):1010–1023. doi: 10.1080/15384101.2015.1007003

Figure 1.

Figure 1.

Assembly of pre-RC onto a plasmid DNA, depending on GAL4 DNA-binding sites. (A) Schematic of pre-RC assembly onto a plasmid DNA, depending on the binding sites. Plasmids attached to magnetic beads were used for the assembly of pre-RC. (B) Binding of GAL4 Cdc6, Mcm2, and Orc2 to the plasmids. Plasmids with various copy numbers of the GAL4 DNA-binding site (6×, 1×, 0×) were attached to the magnetic beads and incubated in a binding buffer with GAL4-Cdc6 on ice for 10 min. After incubation, the bead-coupled plasmids were isolated, washed with a high-salt buffer containing 350 mM NaCl and 100 mM KCl, and incubated in Cdc6-depleted egg extracts (ΔCdc6 LSS) at 23°C for 30 min. The plasmids were then isolated and washed with a low-salt buffer containing 100 mM KCl. Proteins bound to the plasmids were separated by SDS-PAGE and analyzed by immunoblotting. (C) Mcm2 binding to the plasmids, depending on the bound GAL4-Cdc6. The bead-coupled plasmids with or without the DNA-binding sites were pre-incubated in a binding buffer containing GAL4-Cdc6 or GAL4-GST on ice for 10 min. The washed plasmids were then incubated in ΔCdc6 LSS at 23°C for 30 min to assemble pre-RC on the plasmid. Proteins bound to the plasmids were analyzed. (D) Inhibition of Mcm2 binding to the plasmids by geminin. The bead-coupled plasmids bound with GAL4-Cdc6 were incubated in ΔCdc6 LSS in the presence or absence of 1 μM GST-geminin. The plasmids without GAL4-binding sites were used as a negative control (lane 1). (E) ORC-dependent Mcm2 binding to the plasmids bound with GAL4-Cdc6. The bead-coupled plasmids bound with GAL4-Cdc6 were incubated in Cdc6-depleted (ΔCdc6) or Cdc6 and Orc2 double-depleted (ΔOrc2ΔCdc6) LSS at 23°C for 30 min. The proteins in the extracts (extract) and bound to the plasmids (plasmid) were analyzed. (F) High-salt-wash-resistant binding of Mcm2 to the plasmids bound with GAL4-Cdc6. The bead-coupled plasmids incubated in ΔCdc6 LSS were isolated and washed with either the low-salt (100 mM KCl; L) or the high-salt (250 mM KCl; H) buffer after the GAL4 pre-RC was assembled on the plasmids. Proteins bound to the plasmids were analyzed.