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RPA promotes ssDNA-directed synthesis of dsRNA by QDE-1.

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posted on 2010-10-05, 01:54 authored by Heng-Chi Lee, Antti P. Aalto, Qiuying Yang, Shwu-Shin Chang, Guocun Huang, Daniel Fisher, Joonseok Cha, Minna M. Poranen, Dennis H. Bamford, Yi Liu

(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.

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