Selective inactivation of the exonuclease activity of bacteriophage T7 DNA polymerase by in vitro mutagenesis.
The 3' to 5' exonuclease activity of bacteriophage T7 DNA polymerase (gene 5 protein) can be inactivated selectively by reactive oxygen species. Differences in the enzymatic properties between the two forms are exploited to show by a chemical screen that modification of a histidine residue reduces selectively the exonuclease activity. In vitro mutagenesis of the histidine at residue 123, and of the neighboring residues, results in varying reduction of the exonuclease activity, including mutant enzymes that have no detectable exonuclease activity; as a consequence their polymerase activity is increased up to 9-fold. T7 phage containing the mutant genes have a greatly reduced burst size and demonstrate up to a 14-fold increase in the spontaneous mutation rate.
Tabor
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Richardson
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D000595:Amino Acid Sequence; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D005090:Exodeoxyribonucleases / Q000235:genetics*; D005814:Genes, Viral; D006639:Histidine; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D013329:Structure-Activity Relationship; D013604:T-Phages / Q000201:enzymology* / Q000254:growth & development; D014779:Virus Replication
D006639:Histidine; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases