Deoxyribonucleic acid polymerase from Tetrahymena pyriformis. Purification and properties of the major activity in exponentially growing cells.

The major DNA polymerase activity from exponentially growing Tefrahymena pyriformis was isolated and purified approximately l&000-fold relative to the specific activity of DNA polymerase in crude extracts. The enzyme aggregates at low ionic strength. In solutions of ionic strength 0.25 M or greater, the enzyme exists as a single species with an approximate molecular weight of 80,000. DNase activity is associated with polymerase activity throughout all stages of purification. In the presence of MnC12, the enzyme will utilize a polyribonucleotide as template in the presence of an oligodeoxy nucleotide primer. The maximal velocity and K,,, of the enzyme are greater with dTTP than with BrdUTP as substrate when poly[d(A-T)] .poly[d(A-T)] is used as templateprimer. Ethidium bromide is a potent inhibitor of enzyme activity, but inhibition is dependent on concentration of templateprimer and purity of enzyme. This suggests that care must be exercised in comparing different polymerases according to the degree of inhibition of activity by ethidium bromide. Rifamycin SV does not inhibit DNA polymerase from Tefrahymena, but some semisynthetic derivatives of rifamycin SV are potent inhibitors of the enzyme, the most potent being AFf013. DNA polymerase from untreated exponentially growing Tefrahymena appears to be similar in a number of its properties to properties reported for the DNA polymerase induced after treatment of Tefrahymena with ethidium bromide. However, it is not yet known whether or not the enzymes from untreated and from induced cells are the same.