FIGURE 3.

Distinguishing cis and trans FRET. A, duplex substrates and conditions were designed to distinguish cis versus trans IN-induced FRET. One fixed duplex contained only the FRET donor at the 5′ end of the noncleaved strand, as in the standard duplex substrate. A second set of duplexes contained only the acceptor fluorophore at three different positions on the cleaved strand (substrates B, C, and D). These substrates were designed to detect orientation-specific trans FRET. Equal amounts of separately labeled duplexes were first mixed, and then IN was added and the mixture left on ice for 5 min before FRET readings were taken. B, measurement of trans FRET efficiencies between duplexes containing only the acceptor or donor fluorophore and incubated separately with IN on ice for 5 min before mixing. FRET readings were taken immediately thereafter. The total substrate DNA concentration was 50 nm, and the ASV IN to DNA ratio was 4 in all experiments. The experimental schemes are illustrated below each graph with labeling conventions as in Figs. 1 and 2.