Effect of reaction conditions and 3AB on the mutation rate of poliovirus RNA-dependent RNA polymerase in a alpha-complementation assay.
The fidelity of poliovirus RNA-dependent RNA polymerase (3Dpol) was determined using a modified alpha-complementation assay. Several polymerases have been analyzed by this approach allowing comparisons to be drawn. Various conditions including high and low MgCl(2), replacing MgCl(2) with MnCl(2), skewed nucleotide pools, and the presence of poliovirus protein 3AB were analyzed. The assay included RNA synthesis by 3Dpol on an RNA template that coded for a region of the alpha peptide of beta-galactosidase (lacZ-alpha). The product of this reaction was used as a template for a second round of 3Dpol synthesis and the resulting RNA was reverse transcribed to DNA by reverse transcriptase. 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 lacZ-alpha. Although 3AB strongly stimulated 3Dpol synthesis as expected, no change in fidelity was detected. Changes in MgCl(2) also showed little effect. Mutation rates of approximately 9 x 10(-5) (approximately 1 error per 11,000 incorporations) were estimated for these conditions. In contrast, MnCl(2) or skewed nucleotide pools were highly mutagenic resulting in lowered fidelity.
Department of Cell Biology and Molecular Genetics, University of Maryland, Biosciences Research Bldg., College Park, MD 20742, United States. jdestefa@umd.edu
Jeffrey J
JJ
DeStefano
D001483:Base Sequence; D002712:Chlorides / Q000494:pharmacology; D003067:Coenzymes / Q000494:pharmacology; D004269:DNA, Bacterial / Q000096:biosynthesis; D029968:Escherichia coli Proteins / Q000096:biosynthesis; D015636:Magnesium Chloride / Q000494:pharmacology; D017895:Manganese Compounds / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D017955:Poliovirus / Q000201:enzymology*; D012329:RNA, Bacterial / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics*; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D001616:beta-Galactosidase / Q000096:biosynthesis
C520978:3AB protein, poliovirus; D002712:Chlorides; D003067:Coenzymes; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D017895:Manganese Compounds; D008565:Membrane Proteins; D012329:RNA, Bacterial; D017361:Viral Nonstructural Proteins; D015636:Magnesium Chloride; D012324:RNA-Dependent RNA Polymerase; D001616:beta-Galactosidase; C025340:manganese chloride