APHIDICOLIN-RESISTANT CHINESE-HAMSTER OVARY CELLS POSSESS ALTERED DNA-POLYMERASES OF THE ALPHA-FAMILY

DNA polymerases alpha, delta and epsilon were partially purified and c haracterized from a wild type Chinese hamster ovary (CHO) cell line an d from two aphidicolin-resistant mutant CHO cell lines (BR5 and BR5-20 a). The main characteristics of the wild type and mutant DNA polymeras es were compared in order to reveal differences in the properties of t hese enzymes responsible for the. aphidicolin resistance of the mutant cell lines. Pol alpha's of the mutant cells show: (1) in vitro aphidi colin-resistance, (2) 1.5-3-fold lower specific activity than that of the wild type, (3) resistance to cytosine and adenosine arabinofuranos ide 5'-triphosphate (araCTP and araATP), (4) altered resistance to car bonyldiphosphonate (COMDP) and to alkylphenyl nucleotide analogs (buty lphenyl-dGTP and butylanilino-dATP), and (5) lower activity on poly(dA )/oligo(dT) template-primers. These changes in the biochemical propert ies of this enzyme may result from a mutation in pol alpha gene. Pol e psilon 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 fo r 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, t he mutant pol epsilon showed changes in the template-primer preference ; whereas the mutant pol delta was found to have altered sensitivity t o other inhibitors. These results indicate that pol epsilon and pol de lta are also altered as a secondary effect of mutation in the aphidico lin-resistant cells. It is suggested that these altered properties of the DNA pols of the alpha family are responsible for the in vivo aphid icolin resistance of the mutant cells.