Figure 6. Double hexamer formation occurs quickly upon recruitment of the second Mcm2-7 hexamer.

(A) When the two fluorophores (green circle = Dy549, red circle = Dy649) are not associated, excitation of the donor (D ex) will only yield emission from the donor (D em). However when the two fluorophores are in close proximity, we observe acceptor emission (A em) upon D ex, and a weaker D em signal. Wavelengths represent laser excitation and emissions that were monitored.

(B) Representative fluorescence records for experiments using a mixture of Mcm2-7^7SORT549 and Mcm2-7^7SORT649 showing FRET upon arrival of the second Mcm2-7. Red squares highlight when Mcm2-7^7SORT649 associates with DNA (Mcm2-7^7SORT549 is already present), and blue squares highlight when FRET occurs. Images and records of fluorescence intensity for D ex/D em (Mcm2-7^7SORT549), A ex/A em (Mcm2-7^7SORT649), total emission (D ex / (D em + A em), and FRET (D ex/A em) are shown together with calculated E_FRET.

(C) Histogram of E_FRET is plotted for times when a single Mcm2-7^7SORT549 and a single Mcm2-7^7SORT649 are present (blue bars) or when only Mcm2-7^7SORT549 is associated with the DNA (unfilled grey bars). The histogram displays the first ten consecutive E_FRET measurements after arrival of the second Mcm2-7 for 86 DNA molecules (the same number of molecules and time points were used for the control). E_FRET data below −0.5 was excluded from the plot (3/860 signal points and 17/860 control points).

(D) Double-hexamer formation anticipates Cdc6, Cdt1 and ORC release. Survival after the association of the second Mcm2-7 complex of the no-FRET state (green) and of DNA-bound Cdc6^SORT549 (blue), Cdt1^SORT549 (red), and ORC^1SORT549 (black). Mean times ± s.e.m. until FRET increase and ORC, Cdt1, Cdc6 release are reported for comparison.