Extended Data Fig. 1

a, Representative negative-stain EM micrograph and 2D averages of a human MCM loading reaction (∆N proteins) on a short synthetic yeast origin flanked by nucleosomes. b, Benzonase footprinting assay with human (∆N, no ORC6) and yeast MCM loading reactions that were performed at 30 °C for 30 min, and then incubated with benzonase as indicated for 0.5–20 min. Control reactions were incubated for an additional 20 min before addition of benzonase for 0.5 min. c, Comparison of MCM loading reactions (∆N) using protein concentrations used in biochemistry experiments (BC: 90 nM ORC1-5(1∆N), 150 nM ORC6) and in electron microscopy experiments (EM: 120 nM ORC1-5(1∆N), 120 nM ORC6). Other components at standard concentrations (60 nM MCM2-7, 150 nM CDC6∆N, 150 nM CDT1∆N, 4 nM pJY22 plasmid DNA). d, MCM loading was performed using 3xFL or 3x∆N conditions and ORC6 was titrated. ORC1-5 FL and ORC1-5(1∆N) were used at 120 nM. e, Salt stability of the hDH footprint in a buffer containing 500 mM NaCl. MCM was loaded for 30 min and incubated with salt-containing buffers for the indicated time. Reactions were then diluted and treated with benzonase. f, As e, with a buffer containing 750 mM NaCl. g, Salt stability of double hexamers generated using different reaction conditions. MCM was loaded as indicated for 30 min, and then incubated in a buffer containing 500 mM NaCl for 15 min. Reactions were diluted to low salt and treated with benzonase for 0.5 min. The different reaction conditions were tested in the same experiment, run on separate gels and the gels stained individually. h, MCM loading (∆N, no ORC6) was performed on 2,398 bp doubly biotinylated PCR products in the presence of streptavidin. No ori: fragment of pET21a backbone, Ars1: yeast origin from pJY22, Myc ori: human origin at the Myc locus, LamB2 ori: human origin at the Lamin B2 locus. i, As h, using FL + ORC6 reaction conditions. j, Cryo-EM maps of the hOCCM, hOC₁M and yOCCM. Cdt1 is colored in white to highlight the similar binding mode to MCM in the human and yeast complexes. k, Cut-through view of the hOCCM, hOC₁M and yOCCM illustrating the interactions of the MCM WH domains with ORC–CDC6. MCM6 and MCM7 WH domains interact with ORC in all structures, while MCM3 is only visible when CDC6 is present. The MCM4 WH is visible only in yOCCM. l, Cartoon view of the WH interactions described in k. m, Nuclease footprinting assay showing reaction timecourses (∆N, no ORC6) using ATPγS with (top) and without (bottom) addition of geminin at minute 9 after starting the reaction. n, Timecourse of benzonase treatment (0.5–20 min) after a 30-minute reaction using ATPγS (∆N, no ORC6). A control was incubated for an additional 20 min followed by 0.5 min benzonase treatment.