Separation of two DNA polymerase fractions from Tetrahymena cells after excision-repairable damage to DNA

Abstract

Two DNA polymerase activities, A and B, have been separated from ultraviolet- or electron-irradiated Tetrahymena cells and from cells starved for thymine compounds by use of methotrexate plus uridine. Separation was achieved by gel filtration on a Sephadex G-200 column in the presence of 0.5 M NaCl. The first eluted enzyme, Fraction A, was obtained with about the same specific activity as from untreated cells, whereas the specific activity of the second fraction, B, was increased at least 35-fold in response to any one of the above-mentioned treatments of the cells. The increase in activity was dependent on both protein and RNA synthesis. The activities of both fractions depended on the presence of all four deoxyribonucleoside triphosphates, Mg 2+ and primer DNA, and the reaction catalysed by Fraction B proceeded linearly with time for at least 10 h. The optimal salt concentrations for the two enzyme activities differed considerably. Both thymine starvation and irradiation are known to cause excision-repairable damage to Tetrahymena DNA. From the results presented, therefore, it is suggested that the induced enzyme, Fraction B, might represent a DNA repair polymerase. A possible subunit structure of DNA polymerase from Tetrahymena is also discussed.