Supplementary Figure 4: Kinetic analysis of helicase loading with Mcm2-Mcm5 gate–modified Mcm2–7 complexes.
From: Mechanism and timing of Mcm2–7 ring closure during DNA replication origin licensing
(A) Comparison of Cdc6 release times after loading of the first Mcm2–725FRET* or Mcm2–74SNAP. Fraction of fluorophore-labeled Cdc6 (Cdc6SORT549) released after the first Mcm2–725FRET* (red, N = 56) or first Mcm2–74SNAP (the singly-tagged Mcm2–7 used in ref. 8, gray, N = 82) are plotted. Red lines represent the 95% confidence interval (CI) for the release of Cdc6 associated with Mcm2–725FRET*.
(B) Comparison of Cdc6SORT549 release times after loading of the second Mcm2–725FRET* or Mcm2–74SNAP. Fraction of Cdc6 released after the arrival of the second Mcm2–725FRET* (blue, N = 27 molecules) or the second Mcm2–74SNAP (from ref. 8, gray, N = 70 molecules) are plotted. Blue lines represent the 95% CI for the release of Cdc6 associated with the second Mcm2–725FRET*.
(C) Comparison of ORC release times after loading of the second Mcm2–725FRET* or second Mcm2–74SNAP. Fraction of fluorophore-labeled ORC (ORC1SORT549) released after arrival of the second Mcm2–725FRET* (red, N = 25 molecules) or the second Mcm2–74SNAP (from ref. 8, black, N = 98) are plotted. Red lines represent the 95% CI for the ORC release from Mcm2–725FRET*.
(D) Additional analysis of Mcm2–725quench. Record of population average Equench values after initial Mcm2–725quench DNA association (N = 61 molecules, ± s.e.m.). Only first Mcm2–7 associations where the D fluorophore was present for >100s and for which no second Mcm2–7 association was detected within 100s were analyzed. Equench was calculated as a fraction of the fluorescence intensity relative to the first time point of Mcm2–725quench association.
(E) Relative timing of second Mcm2–7 gate closing and initial double-hexamer interactions. At the indicated times the fraction of DNA molecules associated with two Mcm2–7 complexes that show double-hexamer interactions (green; N = 106 molecules, data taken from ref. 8, measured by EFRET between the first and second Mcm2–7) or the fraction of second Mcm2–7 with a closed gate (blue; N = 47 molecules) are plotted. Note that the vertical axis is inverted and that zero on the horizontal axis corresponds to the arrival of the second Mcm2–7. Inset shows mean times after second Mcm2–7 arrival (± 95% CI). Records for gate closure were selected as described for Fig. 2A.
