Supplementary Figure 3: FRET experiments capture the polarity of RPA binding on ssDNA.

(a) & (c) Models of the expected FRET states and the polarity of the Cy5-DBD with respect to the Cy3-DNA. Boxes are color coded to match the traces in the data. Stopped flow experiments were performed by rapidly mixing either RPA-DBD-A^Cy5 or RPA-DBD-D^Cy5 with ssDNA labeled at the (b) 5′ end or (d) 3′ end with Cy3. Cy3 was excited and change in Cy5 emission was measured. The proximity of the Cy3 and Cy5 fluorophores dictate the observed FRET efficiency and results in the enhancement of Cy5 emission. Since RPA binds to ssDNA with a 5′→3′ polarity, when RPA-DBD-A^Cy5 resides close to the 5′Cy3 on DNA, a high FRET state is observed. Similarly, when RPA-DBD-D^Cy5 binds close to the 3′Cy3, a high FRET signal is captured. k_obs values for change in FRET were obtained by fitting the data to a single exponential plus linear equation. Black traces are DNA only (no RPA). Pink and Green traces are experiments with RPA-DBD-A^Cy5 and RPA-DBD-D^Cy5, respectively. The measured rates match well to the rates observed for RPA labeled with MB543 (Fig. 1e, f) suggesting that the ssDNA dependent changes in RPA-MB543 intensity reflect specific DBD-ssDNA interactions.