Further characterization of a poly(rA) · oligo(dT)-dependent activity of multiple DNA polymerase α from calf thymus

DNA polymerase α (EC 2.7.7.7) from calf thymus has been separated into three molecular species, i.e., 10 S DNA polymerase α, 6.5 S DNA polymerase α-1 and 6.5 S DNA polymerase α-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 α and 6.5 S DNA polymerase α-2 were found to copy efficiently poly(rA) · oligo(dT), a template-primer, which was thought to be specific for DNA polymerase γ or β. 6.5 S DNA polymerase α-1, however, could not use the ribopolymer as a template. The poly(rA) · oligo(dT)-dependent activities of DNA polymerase α 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 α, did not inhibit the poly(rA) · oligo(dT)-dependent activity. 2′,3′-DideoxyTTp, a potent inhibitor of DNA polymerase β or γ, slightly inhibited the reactions with poly(rA) · oligo(dT), while it did not inhibit the reactions with activated DNA. The apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for dTTP on poly(rA) · oligo(dT) template were 260 and 70 μM for 10 S α and 6.5 S α-2, respectively; these values were much higher than those obtained on activated DNA template (8–10 μM).