Figure 4. Evidence that Dicer-2 is Processive.

(A) Site-specifically ³²P-radiolabeled 120 bp dsRNAs were used to measure the rate of production of the first, second and fourth siRNAs from the all-RNA end of the substrate; the 32 end of the sense strand contained two deoxynucleotides (red) to block entry of Dicer-2 from the other end (Figure S4A). Production of siRNA was monitored in the presence or absence of ATP using 100 nM dsRNA and 5.4 nM Dicer-2. Values correspond to the mean ± standard deviation from three independent experiments. The data were fit to a single exponential function. In (A) and (B), pink denotes the position of the ³²P-radiolabel. (B) The rate of production of the initial siRNA was measured as in (A), but using a site-specifically ³²P-radiolabeled dsRNA with either a blunt (black) or a 2 nt 3′ overhanging (red) end in the presence or absence of ATP. (C) The rate of production of the initial siRNA was monitored using 100 nM Dicer-2 and 10 nM dsRNA bearing either a blunt (left) or a 2 nt 3′ overhanging (right) end in the presence or absence of ATP or ATPγS. (D) The rate of production of the initial siRNA from a 120 bp blunt ended dsRNA (10 nM) was measured using 100 nM mutant Dicer-2^G31R. (E) The rate of production of the first and fourth siRNAs from a 120 bp blunt ended dsRNA (100 nM) was measured using 100 nM Dicer-2^G31R. Values are mean ± standard deviation for three independent experiments.