Single molecule FRET histograms and time traces measured from RecA filaments formed with ATPγS reveal no direct influence of pH on Cy3—Cy5 dye pair.

(a) A schematic model of RecA filament formed with ATPγS. The dyes are labeled internally with 9 nt separation on the single stranded overhang consisting of polythymine. The distance from the junction to the dye pair is 30 nt and the distance from 3’ end is 22 nt. Double stranded region for surface immobilization is the same as that used in Fig. 1.
(b) Single molecule FRET histograms measured at different pHs. While the bare DNA showed a peak at E∼0.8 (top panel), RecA filaments formed with ATPγS showed a peak at E∼0.4 regardless of pH. Because the dye separation is 9nt, only a stable three monomer binding state between the dyes is expected. A peak at 0 is due to donor-only molecules.
(c-e) Representative single molecule intensity and FRET traces measured at different pH. For each pH condition, two representative traces were shown. Traces in the left panels were chosen to show a single-step photo bleaching event of acceptor at about 35∼40 sec.
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