Fig. 1: Visualization of origin-dependent CMG assembly by electron microscopy.

a, Workflow for the assembly of CMG on a chromatinized origin of replication for electron microscopy (EM) imaging. HSW, high-salt wash; LSW, low-salt wash; NCP, nucleosome core particle. b, Left, 2D averages derived from NS-EM imaging of the CMG assembly reaction. Centre, raw images and right, in silico reconstitution (ReconSil) of the double hexamer (DH) or dCMGE particles on the chromatinized origin of replication. Bottom, representation of the double-hexamer-to-CMG conversion efficiency. c, Measure of inter-nucleosome distance matches the expected length of the ARS1 origin of replication (n = 444 origins for double hexamer; n = 186 origins for dCMGE). Error bars, mean ± s.d. d, Comparison between MCM loading on short DNA containing MH roadblocks. After HSW treatment, equal amounts of loaded MCM helicases are eluted from Strep-TactinXT beads. The black arrowhead indicates MH-bound DNA. For gel source data, see Supplementary Fig. 1. This experiment was performed twice. e, Analysis of the replication products by alkaline agarose gel electrophoresis indicates that short nucleosome- and MH-capped origins can be replicated. For gel source data, see Supplementary Fig. 1. This experiment was performed twice. f, Replication reaction performed as shown in d except on large ARS1 circular DNA of wild-type and mutant MCMs. Mutants include Mcm2 6A, which targets residues that are involved in DNA untwisting; Mcm6 2E, which targets the Mcm6 wedge insertion; and Mcm6 5E, which targets the safety latch. For gel source data, see Supplementary Fig. 1. This experiment was performed twice. g, ReconSil of dCMGE formation on a 6× ARS1 array built from loaded double hexamers. This experiment was performed three times.