Figure 4: Direct measurement of the in vivo unloading time of the β2-sliding clamp during replication.
From: Slow unloading leads to DNA-bound β2-sliding clamp accumulation in live Escherichia coli cells
(a) Illustration of the measurement sequence to image a single β2-clamp unloading event. First a phase-contrast (PH) and pre-activation snapshot are taken, after which molecules are activated only once, and subsequently imaged until foci are no longer visible (b,c) Single PAmCherry–β molecules are visualized by PALM. The sample PH image together with the respective pre-activation and post-activation fluorescence images illustrate that a single PAmCherry–β molecule can successfully be photoactivated. The corresponding pre-activation (red) and post-activation (blue) line profile plots of the single DNA-bound PAmcherry–β molecule. Scale bar, 1.6 μm. (d) A representative example of a montage showing the fluorescence intensity of a PAmCherry-β molecule over time and the corresponding intensity trace of the signal. The single-step disappearance is indicative of a single molecule. (e) On-time distribution for individual PAmCherry-β molecules (n=84) fitted with an exponential (red line), and the distribution for the unloading times corrected for photobleaching (dashed green line). The inset shows the distribution of the fitted unloading time constants over the 106 bootstrapped data sets from which the confidence interval for the unloading time is determined.
