Helicase mutations differentially affect processing of BLT and 3′ovr dsRNA. (A) Schematic of the open-reading frame of Dcr-2. Domains are labeled along with mutations used in this study. (B) Cartoon of the three-dimensional structure of Dcr-2, with subdomains labeled, and arrows pointing to regions of terminus binding sites. (C) Cartoon of dsRNAs used in D, both labeled with 32P on the 5′ end of the sense strand. Modifications that block Dcr-2 binding are indicated on the right side of dsRNAs. (D) PhosphorImage shows a representative 16% denaturing polyacrylamide gel monitoring cleavage of BLT or 3′ovr 52-dsRNAs, as shown in C. Cleavage by Dcr-2, WT, or variants as indicated, was assayed without ATP or with (+) 5 mM ATP. The first three lanes on the left show decade size markers (10 nt lengths) and unreacted dsRNA (1 nM). Lanes 4–15, Dcr-2 150 nM, dsRNA 1 nM. siRNA product is labeled; arrow points to 15 nt product.
