Further studies on calf thymus DNA polymerase .delta. purified to homogeneity by a new procedure

Abstract

DNA polymerase .delta. from calf thymus was purified to apparent homogeneity by a new procedure which utilizes hydrophobic interaction chromatography with phenyl-Sepharose at an early step to separate most of the Ca-dependent protease activity from DNA polymerases .delta. and .alpha.. The purified enzyme migrates as a single protein band on polyacrylamide gel electrophoresis under nondenaturing conditions. The sedimentation coefficient of the enzyme is 7.9S, and the Stokes radius is 53 .ANG.. A MW of 173 kDa [kilodaltons] was calculated for the native enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the homogeneous enzyme reveals 2 polypeptides of 125 and 48 kDa. This subunit structure differs from that of DNA polymerase .delta. prepared by a previous procedure, which was composed of subunits of 60 and 49 kDa, suggesting that the 60-kDa polypeptide may have been derived from the 125-kDa polypeptide during enzyme purification, possibly as the result of cleavage of an unusually sensitive peptide bond. DNA polymerase .delta. is separated from DNA polymerase .alpha. by hydrophobic interaction chromatography on phenyl-Sepharose; DNA polymerase .delta. is eluted at pH 7.2 and DNA polymerase .alpha. at pH 8.5. DNA polymerase .delta. can also be separated from DNA polymerase .alpha. by chromatography on hydroxylapatite; DNA polymerase .alpha. binds to hydroxylapatite in the presence of 0.5 M KCl, whereas DNA polymerase .delta. is eluted at 90 mM KCl. DNA polymerase .delta. is associated with a 3''- to 5''-exonuclease activity, but it is devoid of endonuclease or 5''- to 3''-exonuclease activities. The polymerase activity of DNA polymerase .delta. is inhibited by aphidicolin, as is the 3''- to 5''-exonuclease activity when the substrate is double-stranded DNA. Aphidicolin does not inhibit exonuclease activity on single-stranded DNA. The coordinated inhibition of both polymerase and exonuclease activities of DNA polymerase .delta. by aphidicolin is consistent with the observation that both activities reside on the same protein molecule. The present findings are also consistent with the suggestion that the binding of aphidicolin to the enzyme requires the formation of a template-primer-DNA polymerase complex.