Supplementary Figure 5: Further analysis of Mcm2–725FRET-5RA.
From: Mechanism and timing of Mcm2–7 ring closure during DNA replication origin licensing
(A) Diagram illustrating the ATPase sites formed at the interface between each pair of Mcm2–7 subunits and the location of the Mcm5RA mutation relative to the Mcm2-Mcm5 gate (view is from the C-terminal end of Mcm2–7). The extension from each Mcm subunit represents the arginine finger required for ATP hydrolysis. The cut out of each subunit represents the primary ATP binding site including the Walker A and B motifs.
(B) Evolution of the EFRET distribution for Mcm2–725FRET-5RA associations where both D and A fluorophores were present (N = 33 molecules). The plot is a two-dimensional Gaussian kernel histogram with bandwidths 5.4 s and 0.05 on the time and EFRET axes, respectively. The initial binding of Mcm2–725FRET-5RA is set to time zero.
(C) Two example recordings of labeled Mcm2–74SNAP-mcm5RA/Cdt1SORT549 complexes associating with an individual origin DNA molecule. Both labeled proteins associate simultaneously with the DNA; in one example (left) they persist until the end of the recording (N = 57/109 molecules); in the other they dissociate simultaneously within experimental uncertainty (N = 40/109 molecules).
