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. 2013 Oct 7;110(43):17302–17307. doi: 10.1073/pnas.1311065110

Fig. 3.

Fig. 3.

XerCD–dif–FtsK recombination intermediates. (A) A cartoon of the recombination reaction within substrate II (Fig. S1B). Recombination of this substrate generates a circular DNA product, carrying both fluorophores; its dissociation was assayed by the simultaneous disappearance of both red and green fluorescence emission. (B) Initial synaptic complexes in the absence of FtsK. A representative time and a histogram of FRET efficiency are shown. The mean lifetime of the synapses, τ, is indicated (n = 382; Fig. S3A). (C) Productive recombination events in the presence of FtsK–ATP. Intermediates of recombination were distinguished by their different apparent FRET efficiencies. The following intermediates could be identified: initial synaptic complex (highlighted yellow), XerD–active synaptic complexes, XerD*, and/or XerD HJ (green), and XerC HJ/XerC product (XerC–P) (cyan). Dissociation of the complex upon completion of recombination is indicated in the time trace with an arrow. Color-coded histograms of FRET efficiencies for each step and the mean lifetimes, τ, are shown on the Right (n = 134; Fig. S3A). We note good agreement between the lifetime of the high FRET state between substrates I and II, shown in this figure and in Fig. 2C. The dwell times indicated are for individual transitions, not the entire reaction. To complete the kinetics of the pathway, it is necessary to combine rates from all three substrates. (D) Complexes in the presence of XerCKQD and FtsK. Data were taken with a frame rate of 20 Hz. The complexes were transiently trapped at the HJ intermediate stage, and exhibited a rapid interconversion between E* ≈ 0.22 and ≈ 0.37, suggesting rapid isomerization between XerC–HJ and XerD–HJ. These complexes assembled in the initial conformation (highlighted yellow) and adopted the same conformation before dissociation (purple). Histograms of FRET efficiencies are shown on the Right (n = 84). (E) Complexes in the presence of XerCDKQ and FtsK. The representative time traces display multiple reversible reductions in FRET signal to E* ≈ 0.44, suggesting the transient formation of XerD*. A histogram of FRET efficiencies is shown on the Right (n = 66).