Figure 1. Mcm2-7 hexamers associate with and are loaded on DNA in a one-at-a-time manner.
(A) Schematic for the single-molecule helicase-loading assay. Alexa488-labeled (blue circle) 1.3kb origin-DNAs were coupled to microscope slides. Purified ORC, Cdc6, and Cdt1/Mcm2-7 (at least one fluorescently labeled, Mcm2-7 in this illustration) were incubated with slide-coupled DNA and colocalization of the fluorescently-labeled protein with the DNA was monitored.
(B) Mcm2-7 complexes sequentially associate with origin DNA. Plots display the Mcm2-74SNAP549 fluorescence intensity recorded at two representative DNA molecules. Insets show fluorescence images (4 × 1 s) taken during the sequential association of first (red arrow) and second (blue arrow) Mcm2-7.
(C) Mcm2-7 dwell times on DNA have a multiexponential distribution. Mcm2-7 dwell times were plotted as a histogram. Combined data from first and second Mcm2-7 associations are included; vertical axis represents the number of dwells of the specified duration per s per DNA molecule. Red bars are results from a separate experiment using mutant origin DNA. Inset shows the distribution of Mcm2-7 dwell times on DNA molecules as a semilogarithmic cumulative survival plot; only a portion of the entire plot is shown to emphasize that the distribution has at least two exponential components.
(D) Mcm2-7 associates with DNA one at a time. The number of associations present at standard protein concentrations before a reaction-buffer (top) or high-salt-buffer (0.5 M NaCl; bottom) wash is compared to the number of fluorophores that are detected by photobleaching immediately after the wash.
(E) Two representative traces before and after a high-salt wash and photobleaching. Reactions were washed twice with a high-salt buffer and imaged at higher laser power in the absence of an oxygen scavenging system until all fluorophores were photobleached. Traces of Mcm2-74SNAP549 associations during the reaction (green) are plotted adjacent to photobleaching steps after a high-salt wash (blue).