RPA promotes ssDNA-directed synthesis of dsRNA by QDE-1.
(A) In vitro DdRP assay using circular ssDNA from bacteriophages M13 or φX174 as templates. The nature of the products was characterized by various nuclease treatments and native agarose gel (0.6%) electrophoresis. (B) In vitro DdRP assay using full-length recombinant QDE-1 with various concentrations of the RPA complex. The 175 nt ssDNA template was pre-incubated with RPA before adding QDE-1, and the products were resolved by 6% urea containing polyacrylamide gel. The DNA/RNA hybrid refers to the single-stranded, labeled RNA products of the denatured hybrids. The dsRNA species migrated at higher molecular weight positions in the denaturing gels due to their synthesis by back-priming initiation. (C) The QDE-1 RdRP products with 0 or 70 nM RPA were treated with recombinant Dicer and resolved in 6% (top) or 16% (bottom) urea-containing polyacrylamide gels. The DNA/RNA hybrid refers to the single-stranded, labeled RNA products of the denatured hybrids.