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

The observation that only a subset of the KH mutants were competent for recombination, 205 presumably by a copy-choice mechanism (37), provided the opportunity to determine the extent to 206 which the poly(rU) polymerase activity can predict biological phenotypes. The congruence between 207 the two experiments was remarkable (Fig. 4B) The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/769224 doi: bioRxiv preprint polymerase activity (Fig. 4B) . These results further confirm template switching as the primary 210 mechanism of poly(rU) polymerase activity and validate this assay as a screen for identification of 211 RdRps with deficits in template switching. be identified as the one-nucleotide-extended product (n+1). Addition of the remaining nucleotides in 220 the absence or presence of the acceptor template will yield a strong-stop RNA product. In the presence 221 of a complementary acceptor RNA, the strong-strop RNA product (donor) will be extended, creating a 222 transfer product. 223 We evaluated each KH-containing RdRp derivative in this assay. Substrate and product 224 analysis are presented in Fig. 5C , with the quantitation of the transfer product relative to WT presented 225 in Fig. 5D . KH RdRp was defective in this assay as well, as transfer product was not detected (Fig. 226 5C). Importantly, the failure to transfer was not a reflection of the inability to assemble or produce 227 strong-stop donor RNA (Fig. 5C) . In this assay, PS-KH RdRp outperformed IF-KH RdRp, and PS-KH 228 and IF-PS-KH RdRp were essentially identical in activity. 229 We conclude that copy-choice and forced-copy-choice recombination use distinct mechanisms, The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/769224 doi: bioRxiv preprint