(a) A schematic of the molecular events during helicase loading. The ORC and Mcm2-7 proteins are labeled at their C-termini (C) which form an interface during first Mcm2-7 recruitment (Yuan et al., 2017). ORC5C-549 is labeled with a donor fluorophore (D; green circles) and Mcm2-72C-649 is labeled with an acceptor fluorophore (A; red circles). The associated fluorescence images show single frames of ORC, Mcm2-7, and raw FRET fluorescence spots at a single DNA molecule in green, red, and blue outlines, respectively. We use gray and yellow coloring to indicate the first and second Mcm2-7 hexamers, respectively. (b) Schematic of DNA-bound ORC5C-549 and Mcm2-72C-649 proteins. When ORC5C-549 and Mcm2-72C-649 are not associated, excitation of the donor (Dex) only yields emission from the donor fluorophore (Dem). However, when ORC5C-549 and Mcm2-72C-649 are in proximity, we observe emission from the acceptor (Aem) on donor excitation due to FRET, and corresponding lower emission from the donor fluorophore (Dem). (c) A representative trace showing ORC and Mcm2-7 associations with DNA and ORC–Mcm2-7 (OM) interactions during helicase loading. Donor-excited fluorescence record shows ORC5C-549 association (green: Dex, Dem panel) and acceptor-excited fluorescence record shows Mcm2-72C-649 association (red: Aex, Aem panel). Gray arrows link to the corresponding molecular events shown in (a). Interaction between ORC and Mcm2-7 decreases the distance between the donor and acceptor fluorophores and results in increased apparent FRET efficiency (EFRET, blue: Dex, [Aem/ (Aem + Dem)] panel). EFRET values are calculated using donor-excited emission from the donor and acceptor fluorophores (see Materials and methods) and they are therefore shown only at times when both a donor and acceptor fluorophore are present on the DNA molecule. The black: Dex, (Aem + Dem) panel shows donor-excited total emission (see Figure 1—figure supplement 3). An objective image-analysis algorithm (Friedman and Gelles, 2015) detects a spot of fluorescence at time points shown in green, red, and black on the time records. Gray, gray-dash, and yellow highlight three 10-s time intervals referenced in (d). A.U., arbitrary units. Concentrations of labeled proteins in the reaction are 0.5 nM ORC5C-549, 15 nM Cdt1–Mcm2-72C-649. Figure 1—figure supplement 2a shows additional records. (d) Histogram plots of EFRET values for 106 single-ORC-mediated, double-hexamer formation events during three 10-s time intervals: immediately after the first (top) or second Mcm2-7 (bottom) arrives or before the second Mcm2-7 arrives (middle). Examples of these intervals are indicated in (c) with gray, yellow, and gray-dash, respectively. The two dashed lines indicate EFRET values of 0 and 1. NDH, number of double-hexamer formation events and nt, number of signal points. Rare EFRET values below −2 and above +2 were excluded (2/428, 13/428, and 1/428 signal points from the top, middle, and bottom histograms, respectively).