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. 2011 Apr 2;68(22):3741–3756. doi: 10.1007/s00018-011-0675-9

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Fig. 2 — Human Orc6 is required for pre-RC formation but not for ORC DNA-binding. a Left Silver-stained SDS-PAGE of affinity-purified human Orc1–5 after baculovirus mediated overexpression. Right HsOrc1–HsOrc5 and bacterially expressed HsOrc6 were employed to monitor DNA binding activities in EMSA experiments; 150 fmol of a Cy5-labeled double-stranded AT-rich oligonucleotide [41] was incubated with 5 pmol Orc6 (lanes 2–4), 1 pmol Orc1–Orc5 (lanes 8–10), and Orc1–Orc6 (lanes 5–7). Increasing amounts of poly(dI-dC) were used as competitor (lanes 3, 6, and 9 50 ng, lanes 4, 7, and 10 250 ng). Control lane 1 no protein. b Outline of the cell-free plasmid-binding assay to study the assembly of pre-RCs in vitro. Biotinylated supercoiled pEPI-UPR was immobilized to paramagnetic beads and incubated with nuclear extract [4]. Proteins bound to the beads were detected by immunoblotting. c ATP is required for Mcm2–Mcm7 protein loading and induces phosphorylation of DNA-bound Cdc6. Lane 1 beads only control. The binding reaction was performed with DNA without adding further ATP (lane 2) or in the presence of an ATP-regenerating system (lane 3). The putative phosphorylation of Cdc6 was addressed in an independent experiment (lanes 4–6) by λ-phosphatase treatment (PPase, lane 6) of the bead-bound material before the SDS-PAGE. A λ-PPase-untreated reaction is shown for comparison (lane 5). Lane 4 beads only control. d Depletion of ORC subunits reduces pre-RC formation. Orc2- but not Orc6-specific antibodies co-deplete Orc1–Orc5 proteins (lanes 1–3). Immobilized plasmid DNA was incubated with nuclear extract either depleted with control (α-IgG, lanes 6, 10), Orc2- (lane 7) or Orc6-specific antibodies (lane 11). For complementation, 120 ng of recombinant Orc6 protein was added to the Orc6-depleted nuclear extract (lane 12). Lanes 5–7 DNA binding of Orc1, 2, 4, 6 and Cdc6 was quantified in relation to the amount of protein bound to DNA (lane 5). Lanes 9–12 Plasmid binding of Orc1, -2, -4 and -6 proteins, as well as Mcm7 and Cdc6 was monitored and quantified in relation to lane 9. All experiments were performed in the presence of 1 mM ATP and an ATP regenerating system

Fig. 2 — Human Orc6 is required for pre-RC formation but not for ORC DNA-binding. a Left Silver-stained SDS-PAGE of affinity-purified human Orc1–5 after baculovirus mediated overexpression. Right HsOrc1–HsOrc5 and bacterially expressed HsOrc6 were employed to monitor DNA binding activities in EMSA experiments; 150 fmol of a Cy5-labeled double-stranded AT-rich oligonucleotide [41] was incubated with 5 pmol Orc6 (lanes 2–4), 1 pmol Orc1–Orc5 (lanes 8–10), and Orc1–Orc6 (lanes 5–7). Increasing amounts of poly(dI-dC) were used as competitor (lanes 3, 6, and 9 50 ng, lanes 4, 7, and 10 250 ng). Control lane 1 no protein. b Outline of the cell-free plasmid-binding assay to study the assembly of pre-RCs in vitro. Biotinylated supercoiled pEPI-UPR was immobilized to paramagnetic beads and incubated with nuclear extract [4]. Proteins bound to the beads were detected by immunoblotting. c ATP is required for Mcm2–Mcm7 protein loading and induces phosphorylation of DNA-bound Cdc6. Lane 1 beads only control. The binding reaction was performed with DNA without adding further ATP (lane 2) or in the presence of an ATP-regenerating system (lane 3). The putative phosphorylation of Cdc6 was addressed in an independent experiment (lanes 4–6) by λ-phosphatase treatment (PPase, lane 6) of the bead-bound material before the SDS-PAGE. A λ-PPase-untreated reaction is shown for comparison (lane 5). Lane 4 beads only control. d Depletion of ORC subunits reduces pre-RC formation. Orc2- but not Orc6-specific antibodies co-deplete Orc1–Orc5 proteins (lanes 1–3). Immobilized plasmid DNA was incubated with nuclear extract either depleted with control (α-IgG, lanes 6, 10), Orc2- (lane 7) or Orc6-specific antibodies (lane 11). For complementation, 120 ng of recombinant Orc6 protein was added to the Orc6-depleted nuclear extract (lane 12). Lanes 5–7 DNA binding of Orc1, 2, 4, 6 and Cdc6 was quantified in relation to the amount of protein bound to DNA (lane 5). Lanes 9–12 Plasmid binding of Orc1, -2, -4 and -6 proteins, as well as Mcm7 and Cdc6 was monitored and quantified in relation to lane 9. All experiments were performed in the presence of 1 mM ATP and an ATP regenerating system