RNA-dependent RNA polymerase speed and fidelity are not the only determinants of the mechanism or efficiency of recombination

Hyejeong Kim; Victor D. Ellis; Andrew Woodman; Yan Zhao; Jamie J. Arnold; Craig E. Cameron

After a 24-h incubation at 37 °C, virus was isolated and used for plaque assay. Indicated is the titer of 582 virus recovered in the presence of ribavirin normalized to that recovered in the absence of ribavirin. The heteropolymeric, symmetrical, primed-template substrate (sym/sub) has been used to establish an assay for forced-copy-choice recombination in vitro (22, 38). RdRp assembles on sym/sub. ATP is added and incorporated to yield a stable elongation complex. It is this elongation complex that is monitored for extension and transfer. Transfer is strictly dependent on the presence of an acceptor RNA with 5 complementarity to the 3'-end of donor RNA. (B) Schematic of the experimental design is indicated. Products in boxes are those observed and monitored by denaturing polyacrylamide gel electrophoresis. (C) Reaction products were resolved by electrophoresis and detected by phosphorimaging. The only regions of the gel with bands are shown; these correspond to the sym/sub primer, one-nucleotide-extended primer (n+1), four-nucleotides-extended product (strong stop) and non-templated addition of nucleotides to that product, and the transfer product. (D) Transfer products were quantified and are expressed as