RESUMO
The fidelity of poliovirus RNA-dependent RNA polymerase (3D(pol)) was determined using a system based on the fidelity of synthesis of the alpha-lac gene which codes for a subunit of beta-galactosidase. Synthesis products are screened for mutations by an alpha-complementation assay, in which the protein product from alpha-lac is used in trans to complement beta-galactosidase activity in bacteria that do not express alpha-Lac. Several polymerases 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 for the N-terminal region of alpha-Lac. The product of this reaction was used 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 bacteria were screened for beta-galactosidase activity by blue-white phenotype analysis with white or faint blue colonies scored as errors made during synthesis on alpha-lac. Results showed a mutation rate for 3D(pol) corresponding to approximately 4.5x10(-4) errors per base (one error in approximately 2200 bases). Analysis of mutations showed that base substitutions occurred with greater frequency than deletions and insertions.
