Further characterization of a poly(rA) . oligo(dT)-dependent activity of multiple DNA polymerase alpha from calf thymus.
DNA polymerase alpha (EC 2.7.7.7) from calf thymus has been separated into three molecular species, i.e., 10 S DNA polymerase alpha, 6.5 S DNA polymerase alpha-1 and 6.5 S DNA polymerase alpha-2 (Masaki, S. and Yoshida, S. (1978) Biochim, Biophys. Acta 531, 74-88; Yoshida, S., Yamada, M., Masaki S. and Seneyoshi, M. (1979) Cancer Res. 39, 3955-3958). Among these three, 10 S DNA polymerase alpha and 6.5 S DNA polymerase alpha-2 were found to copy efficiently poly(rA) . oligo(dT), a template-primer, which was thought to be specific for DNA polymerase gamma or beta. 6.5 S DNA polymerase alpha-1, however, could not use the ribopolymer as a template. The poly(rA) . oligo(dT)-dependent activities of DNA polymerase alpha species differed markedly from those with activated calf thymus DNA in sensitivity to various reagents: the former was inhibited more than 80% by 80 mM KCl, while the latter was stimulated somewhat. Furthermore, aphidicolin, a specific inhibitor of DNA polymerase alpha, did not inhibit the poly(rA) . oligo(dT)-dependent activity. 2',3'-DideoxyTTP, a potent inhibitor of DNA polymerase beta or gamma, slightly inhibited the reactions with poly(rA) . oligo(dT), while it did not inhibit the reactions with activated DNA. The apparent Km values for dTTP on poly(rA) . oligo(dT) template were 260 and 70 microM for 10 S alpha and 6.5 S alpha-2, respectively; these values were much higher than those obtained on activated DNA template (8-10 microM).
D000818:Animals; D002417:Cattle; D004257:DNA Polymerase II / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D054306:Dideoxynucleotides; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D009841:Oligonucleotides / Q000494:pharmacology*; D011061:Poly A / Q000494:pharmacology*; D011189:Potassium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D013942:Thymine Nucleotides; D013950:Thymus Gland / Q000201:enzymology*
D054306:Dideoxynucleotides; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D013942:Thymine Nucleotides; D011061:Poly A; C018368:poly(rA).oligo(dT); D011189:Potassium Chloride; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase; C017216:2',3'-dideoxythymidine triphosphate