Determination of the mutation rate of poliovirus RNA-dependent RNA polymerase

The fidelity of poliovirus RNA-dependent RNA polymerase (3D(pol)) was deter mined using a system based on the fidelity of synthesis of the alpha -lac g ene which codes for a subunit of beta -galactosidase. Synthesis products ar e screened for mutations by an alpha -complementation assay, in which the p rotein product from alpha -lac is used in trans to complement beta -galacto sidase activity in bacteria that do not express alpha -Lac. Several polymer ases have been analyzed by this approach allowing comparisons to be drawn. The assay included RNA synthesis by 3D(pol) on an RNA template that coded f or the N-terminal region of alpha -Lac. The product of this reaction was us ed as a template for a second round of 3D(pol) synthesis and the resulting RNA was reverse transcribed to DNA by MMLV-RT. The DNA was amplified by PCR and inserted into a vector used to transform Escherichia coli. The bacteri a were screened for beta -galactosidase activity by blue-white phenotype an alysis with white or faint blue colonies scored as errors made during synth esis on alpha -lac. Results showed a mutation rate for 3D(pol) correspondin g to approximate to 4.5 x 10(-4) errors per base (one error in approximate to 2200 bases). Analysis of mutations showed that base substitutions occurr ed with greater frequency than deletions and insertions. (C) 2001 Elsevier Science B.V. All rights reserved.