Studies on Deoxyribonucleic Acid Polymerase from Ehrlich Ascites Tumor Cells

DNA polymerase obtained from Ehrlich ascites tumor cells by hypotonic shock has been purified by a procedure utilizing acid precipitation, fractionation on diethylaminoethyl cellulose, phosphocellulose, hydroxylapatite, and Sephadex G-100, resulting in a purification of the order of 300-fold with respect to the activity of the initial extract. The purified polymerase was free of endonuclease but contained an exonuclease whose activity was proportional to the polymerase activity throughout the purification. Exonuclease activity, as reflected by loss of template, was not evident under conditions of synthesis, but was easily detected in the absence of DNA synthesis. The enzyme was inactivated by heat in the absence of DNA. In the presence of activated DNA, a transitory enhancement of activity was observed upon heating. Sucrose density gradient centrifugation in the presence of native DNA showed poor affinity of the enzyme for native DNA. All four deoxyribonucleoside triphosphates and magnesium ions were required for activity. Optimal activity was found at pH 7.4, with 7 mm magnesium, 50 mm potassium phosphate, and with less than 120 µm eq of template. The properties of the enzyme suggest that it is neither mitochondrial in origin nor chromatin-bound. It is found both in nuclear and in cytoplasmic fractions of cell homogenates.