A strong mutator effect caused by an amino acid change in the alpha subunit of DNA polymerase III of Escherichia coli.
Most potent mutators heretofore detected in Escherichia coli are associated with defects in epsilon subunit of DNA polymerase III, encoded by the dnaQ gene. To elucidate the role of the alpha subunit, the catalytic subunit of the polymerase, in maintaining the high fidelity of DNA replication, we isolated a mutator mutant, the mutation (dnaE173) of which resides on the dnaE gene, encoding the alpha subunit. The dnaE173 mutant was unable to grow in salt-free L broth at temperatures exceeding 44.5 degrees C and exhibited an increased frequency of spontaneous mutations, 1,000 to 10,000-fold the wild type level, at permissive temperatures. The mutator effect of dnaE173 mutation is dominant over the wild type allele. These phenotypes are caused by a single base substitution, resulting in one amino acid change, Glu612 (GAA)----Lys(AAA), in the alpha subunit molecule. DNA polymerase III purified from the dnaE173 mutant contained both alpha and epsilon subunits, in a normal molar ratio. We found no differences between wild type and mutant polymerases in the Vmax, thermolabilities, and salt sensitivities. However, the apparent Km for the substrate nucleotide of the mutant polymerase was 1/6 of that determined with the wild type polymerase. Although the mutant polymerase retained a normal level of 3'----5' exonuclease activity, the proofreading capacity determined by "turnover assay" was significantly lower in the mutant polymerase, as compared with findings in the normal enzyme. It seems likely that the enhanced mutability in the dnaE173 strain results from, at least in part, a defect in the editing function of DNA polymerase III, and further suggests that a portion of the alpha subunit in which the amino acid change resides may be important for the proper setting of the two subunits at the replication fork so as to facilitate efficient editing during the DNA replication.
Maki
H
H
Department of Biochemistry, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Mo
J Y
JY
Sekiguchi
M
M
D000483:Alleles; D000596:Amino Acids / Q000235:genetics*; D002678:Chimera; D002850:Chromatography, Gel; D004258:DNA Polymerase III / Q000235:genetics*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D008040:Genetic Linkage; D009154:Mutation
D000596:Amino Acids; D004269:DNA, Bacterial; D004258:DNA Polymerase III