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

Lys-359 protonates the pyrophosphate leaving group during nucleotidyl transfer (24, 42). The 250 K359H substitution will change the efficiency of that protonation event for two reasons. First, the pKa 251 value of histidine is substantially lower than lysine and will therefore not be protonated to the same 252 extent. Second, the distance between the nucleotide phosphates and the imidazole ring of the histidine 253 will be greater than that of the amino group of lysine. It is likely that the distance is more of an issue, 254 because K359R PV is stable and K359R RdRp exhibits the same reduction in catalytic efficiency as 255 . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 recombination phenotype (Fig. 3B ) may suggest that forced-copy-choice recombination contributes to 302 recombination in cells as well. 303 The current narrative of the characterized PV mutants impaired for recombination is that 304 recombination acts as a mechanism to purge deleterious mutations in the viral population (45-47). Our conserved residues capable of perturbing conserved, biochemical function (6, 8, 10). As a field, we 316 have focused on RdRp fidelity (6) but have come to the realization that RdRp speed should not be 317 ignored (17). This study shows that speed, fidelity, and undefined biochemical properties of the RdRp 318 exist and contribute to both the mechanism and efficiency of recombination (Fig. 6) . This inextricable 319 connection of the myriad biochemical properties of the RdRp precludes attribution of a single 320 biochemical property to viral fitness, virulence, and pathogenesis (Fig. 6) . The field will be served best The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 and treated with 2U DNAse Turbo (ThermoFisher) to remove residual DNA template. The RNA 348 transcripts were purified using RNeasy Mini Kit (Qiagen) before spectrophotometric quantification.