(A) Schematic of the single-molecule helicase-loading assay. Alexa-Fluor-488-labeled (blue circle) 1.3 kb origin DNAs were coupled to a passivated microscope slide. Mcm2-7 was fluorescently labeled with donor (Dyomic-549, green circle) or acceptor (Dyomic-649, red circle) fluorophores. Purified ORC, Cdc6, and Cdt1/Mcm2–7 were incubated with slide-coupled DNA. Colocalization of fluorescently-labeled proteins with the DNA and any associated FRET signal were monitored. (B) Wild-type Mcm2-7 forms long-lasting FRET signals. Representative fluorescence records for an experiment using a 1:1 mixture of wild-type donor- and acceptor-labeled Mcm2-7 showed FRET after arrival of the second Mcm2–7. Records of fluorescence intensity for (i) acceptor excitation; acceptor emission (Dyomic-649-labeled Mcm2-7, red arrow marks arrival of acceptor-labeled Mcm2-7), (ii) donor excitation; donor emission (Dyomic-549-labeled Mcm2-7, green arrow marks arrival of donor-labeled Mcm2-7) and FRET (donor excitation; acceptor emission, blue arrow marks initiation of FRET), (iii) total emission (donor excitation; donor emission + acceptor emission), and (iv) calculated EFRET are shown. Black arrows indicate both donor and acceptor release due to the double hexamer sliding off the end of the DNA. (C) Representative fluorescence records for experiments using Mcm2-74-178A (labels and arrows as in B) show a short-lived FRET signal. (D) (i) Time evolution of the EFRET distribution for 89 wild-type Mcm2-7 complexes. Only complexes with one donor-labeled and one acceptor-labeled Mcm2-7 were selected. EFRET values were measured only after arrival of the second Mcm2-7, which was taken to be time zero in each record. The plot is a two-dimensional histogram (see Materials and Methods) with Nt = 2688 measurements within the time and EFRET range shown. (ii-iv) Histograms (probability density ± S.E.) of EFRET values recorded during the indicated time intervals after association of the second Mcm2–7 with origin DNA (black bar indicates possible intermediate EFRET state). EFRET values were globally fit to the sum (dashed cyan curves) of two Gaussians (red curves) constrained to have the same peak positions and widths at all times. (E) Analyses analogous to (D) for the DoHM mutant (114 complexes; Nt = 2584 in range shown in (i). For (ii) - (iv) in (D, E), fit parameters and numbers of observations are reported in Supplementary file 1 - table 1.