Figure 4.

TWINKLE interacts with both the displaced-strand trap and the homologous 3′-tail strand to catalyze the strand-exchange reaction. (A) Fluorescence anisotropy competition assay to assess the binding of Twinkle to morpholino oligo. A complex of Twinkle (represented by blue ring) and ssDNA labeled with fluorescein (represented by black line with ‘F’ at one end in the cartoon) was formed first by titrating the labeled ssDNA with increasing TWINKLE, then the complex was titrated either with increasing concentrations of unlabeled ssDNA or the morpholino oligo and fluorescence anisotropy was measured in each case. A drop in fluorescence anisotropy indicating the ‘falling-off’ of TWINKLE from the pre-bound fluorescein-labeled ssDNA was observed only with ssDNA and not with the morpholino oligo. (B) Gel image shows the time course of unwinding the DNA/morpholino hybrid fork with 18-bp duplex region (left) or the DNA/morpholino 5'-overhang DNA with 25-bp duplex region (right). Quantitation shows about 3% unwinding in each case. (C) DNA unwinding in the presence of the morpholino oligo as trap. Kinetics of 5′-tail strand release from the 25-bp fork DNA in the presence of ssDNA 25-nt trap (0.045 ± 0.003 strand/min), ssDNA 60-nt trap (0.06 ± 0.006 strand/min) and morpholino oligo trap. The 60-nt trap is the unlabeled 5′ strand of the forked-substrate DNA.