Fig. 5. Orc6-CBN is required for CDK-mediated inhibition of Mcm7 recruitment to ScORC·DNA·Cdc6.
a Experimental workflow of Mcm7 recruitment assay. Docking of Orc6-CBN onto the ORC·Cdc6 ring is predicted to prevent binding of Mcm7. b Binding of ScMcm7 to ScORC·DNA·Cdc6 is increased after dephosphorylation and decreased upon CDK phosphorylation of ORC. Association of increasing amounts of Mcm7 with the initiator·co-loader was assessed in amylose pulldowns using ORC with MBP-tagged Orc4 as bait. c–e Deletion of Orc6-CBN abrogates the phosphorylation-dependent regulation of ScMcm7 binding to ScORC·DNA·Cdc6. Coomassie-stained SDS-PAGE gels of elutions from amylose pulldowns are shown in (b–e). The mean and standard error of the mean of ScMcm7 band intensities (n = 4 independent recruitment assays) are plotted in (c–e). Note that Mcm7 migrates as a doublet and that Orc2 and the unstructured linker region of Orc6ΔCBN are prone to proteolytic cleavage during expression and purification. Asterisks mark degradation products of ORC subunits that are stably integrated into ORC. In e, a higher percentage gel was used to visualize Orc6ΔCBN/Δlinker due to its low molecular weight. SDS-PAGE gels of inputs from b–e are included in Supplementary Fig. 12. Source data are provided as a Source Data file.