Figure 3.

FRET from synapsis in cis. Equal volumes of FokI endonuclease and a solution of the requisite DNA(s) were mixed by stopped-flow to give reactions in C-buffer at 20°C containing DNA at a total concentration of 25 nM and FokI at 50 nM. Excitation was at 545 nm and the change in emission (a.u.) at >645 nm recorded over time. The black, red and blue traces each come from reactions with the DNA molecule(s) indicated in the right-hand panel. The DNA constructs were 260 bp long and contained two FokI sites (yellow arrowheads, marking their directionality) 30 bp from each end, in inverted or in directly repeated orientations. The DNA was labelled with Ax546 (cyan circle) at one end and with Ax647 (mauve circles) at either the opposite or the same end. The DNA substrate(s) that gave rise to the black, red and blue traces are depicted next to the squiggle in the same colour. The schemes below each set of DNA substrate(s) show the structures formed by parallel and anti-parallel synapses of the FokI sites within that set: the relative locations of the Ax546 and Ax647 dyes are indicated. (For the set of substrates in the blue record, only one of the many possible synapses is shown, a parallel assembly).