Figure 5. A single ORC complex directs recruitment and loading of the first and second Mcm2-7 hexamer.

(A) Representative fluorescence intensity record for ORC^1SORT549 and Mcm2-7^4SNAPJF646 at an origin-DNA location. Association of first and second Mcm2-7 are marked with red and blue arrows, respectively.

(B) A single ORC complex directs recruitment of two hexamers. The fraction (± s.e.) of DNA molecules observed to have zero, one or two ORC fluorophores bound when the second Mcm2-7 was recruited, is plotted (bars) together with the predicted number of associated fluorophores (red and blue squares) of different models (see Fig. S5A).

(C) ORC is released rapidly after recruitment of the second Mcm2-7 hexamer. Histograms showing the time between the association of the second Mcm2-7 and ORC release (top), or association of the first Mcm2-7 and ORC release (bottom).

(D) Release of Cdc6^SORT549 (blue), Cdt1^SORT549 (red), and ORC^1SORT549 (black) after the association of the second Mcm2-7^4SNAPJF646 complex is plotted as a survival function. There are two ORC molecules that associate for >400s (1033.8s and 709.6s) that are not shown and disproportionately affect the mean dwell time. Grey lines represent a 95% confidence interval for the ORC dataset showing that there is no significant difference between Cdt1 and ORC release time distributions. Numbers in parentheses represent the mean release times ± s.e.m.