title: Aphidicolin-resistant Chinese hamster ovary cells possess altered DNA polymerases of the alpha-family.
abstract: DNA polymerases alpha, delta and epsilon were partially purified and characterized from a wild type Chinese hamster ovary (CHO) cell line and from two aphidicolin-resistant mutant CHO cell lines (BR5 and BR5-20a). The main characteristics of the wild type and mutant DNA polymerases were compared in order to reveal differences in the properties of these enzymes responsible for the aphidicolin resistance of the mutant cell lines. Pol alpha's of the mutant cells show: (1) in vitro aphidicolin-resistance, (2) 1.5-3-fold lower specific activity than that of the wild type, (3) resistance to cytosine and adenosine arabinofuranoside 5'-triphosphate (araCTP and araATP), (4) altered resistance to carbonyldiphosphonate (COMDP) and to alkylphenyl nucleotide analogs (butylphenyl-dGTP and butylanilino-dATP), and (5) lower activity on poly(dA)/oligo(dT) template-primers. These changes in the biochemical properties of this enzyme may result from a mutation in pol alpha gene. Pol epsilon and delta of the mutant cells did not differ from the wild type enzymes with respect to aphidicolin resistance. However, the specific activities of these mutant enzymes were much higher (1.5 to 8-fold for pol epsilon and 4 to 20-fold for pol delta) in comparison to that of the wild type enzymes. Also in comparison to the wild type enzymes, the mutant pol epsilon showed changes in the template-primer preference; whereas the mutant pol delta was found to have altered sensitivity to other inhibitors. These results indicate that pol epsilon and pol delta are also altered as a secondary effect of mutation in the aphidicolin-resistant cells. It is suggested that these altered properties of the DNA pols of the alpha family are responsible for the in vivo aphidicolin resistance of the mutant cells.
authors.affiliation: Department of Biological Sciences, University of South Carolina, Columbia 29208.
authors.forename: Z
authors.initials: Z
authors.lastname: Fehér
authors.forename: N C
authors.initials: NC
authors.lastname: Mishra
mesh_terms: D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology*; D001085:Arabinofuranosylcytosine Triphosphate / Q000494:pharmacology; D016466:CHO Cells; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000378:metabolism; D004257:DNA Polymerase II / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D009154:Mutation; D013698:Templates, Genetic; D001084:Vidarabine Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D001084:Vidarabine Phosphate; D001085:Arabinofuranosylcytosine Triphosphate; C000101:ara-ATP; D016590:Aphidicolin; D004247:DNA; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase