Mammalian mutator mutant with an aphidicolin-resistant DNA polymerase alpha.
The Chinese hamster V79 cell mutant aphr-4-2, selected for its resistance to aphidicolin, a specific inhibitor of DNA polymerase alpha (DNA nucleotidyltransferase, EC 2.7.7.7), is characterized by slow growth, UV sensitivity, and hypersensitivity to UV-induced mutation. DNA polymerase alpha has been purified from mitochondria-free crude extracts of the mutant and its parental wild-type cells by sequential column chromatography on DEAE-cellulose and phosphocellulose. The major DNA polymerase activity from both cell lines was found to have characteristics of the alpha-type polymerase: sensitivity to 0.2 M KCl, resistance to heat denaturation (45 degrees C for 15 min), an apparent Km of 5 microM for dATP, and an ability to copy poly(dT)X(rA)10 but not poly(rA)X(dT)12. The crude extracts and purified DNA polymerase alpha from the mutant cells are not inhibited by aphidicolin (greater than 0.6 microM). The apparent Km for dCTP with DNA polymerase alpha is 1.0 +/- 0.4 microM (mean +/- SD) for the mutant enzyme. The polymerase from the parental cells, similarly purified, is sensitive to aphidicolin and has an apparent Km for dCTP of 10 +/- 4 microM. The spontaneous mutation rate (per cell per division), determined by fluctuation analysis at the Na+/K+-ATPase (EC 3.6.1.8) locus, is higher for mutant cells (42-73 x 10(-8)) than for parental cells (3-16 x 10(-8)). These data suggest a mechanism for aphidicolin resistance of the mutant--i.e., a decrease in the Km for dCTP. The results also indicate that an altered DNA polymerase alpha may be intrinsically mutagenic during normal semiconservative replicative as well as during UV-induced repair syntheses.
D000818:Animals; D016590:Aphidicolin; D002478:Cells, Cultured; D006224:Cricetinae; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D003854:Deoxyribonucleotides / Q000378:metabolism; D004224:Diterpenes / Q000494:pharmacology*; D004351:Drug Resistance; D007700:Kinetics; D009154:Mutation; D019384:Nucleic Acid Synthesis Inhibitors
D003854:Deoxyribonucleotides; D004224:Diterpenes; D019384:Nucleic Acid Synthesis Inhibitors; D016590:Aphidicolin; D004259:DNA-Directed DNA Polymerase