Fig. 2: Comparative analysis of double-hexamer formation and Cdt1 interaction with MCM2-7 using purified yeast and human proteins.
Assembled pre-RC reactions were subjected to washes with a sodium chloride gradient ranging from 0 to 500 mM for (a) yeast and (b) human. SDS-PAGE gels are representative of two independent biological replicates. c Negative stain EM 2D class averages generated with CryoSPARC carried out on pre-RC assay reactions washed with 300 mM NaCl from yeast and human. The percentage numbers represent the proportion of the total particles in each class of either double hexamer (DH) or single hexamer (SH). hMCM2-7 DH formation was more variable than yMcm2-7 DH-formation, suggesting additional regulation mechanisms exist. Mass photometry on (d) yMCM2-7 and yCdt1 and (e) hMCM2-7 and hCDT1ΔN, was carried out in solution with a two-fold excess of CDT1. Only yeast proteins form a complex in solution, as detected by an upshift (marked with an asterisk) in the yMCM2-7 mass peak that is proportional to the mass of yCdt1. f Pre-RC assay under low salt conditions was carried out in the absence and presence of hCDT1ΔN, highlighting that initial hMCM2-7 recruitment occurs without hCDT1. SDS-PAGE gel is representative of three independent biological replicates. Source data are provided as a Source Data file.
