Purification and characterization of a deoxyribonucleic acid polymerase from rat liver.

A highly pursed DNA-polymerase has been isolated from normal and regenerating rat liver by differential ultracentrifugation, acid precipitation, ammonium sulfate precipitation, and chromatography on phosphocellulose and on DNAcellulose. The molecular weight of the enzyme is estimated to be 49,000. Enzyme purified in this manner has no detectable terminal transferase, exonuclease, and endonuclease activities. Polymerase activity is absolutely dependent upon the addition of DNA and is maximal in the presence of all four deoxyribonucleoside triphosphates. The pH optimum of the enzyme is 7.7 in Tris-HCl buffer, and the optimum Mg Z+ concentration is 10 mm. Enzyme activity is inhibited 50 % by 0.03 M ammonium sulfate and 94 % by 3 X lop4 M p-chloromercuribenzoate, and is not inhibited by 10 pM ethidium bromide. Synthesis proceeds for a longer period of time in the presence of 0.2 M KCl. Neither native DNA nor denatured DNA serves effectively as a template for the enzyme. Native DNA which has been partially digested with pancreatic DNase, poly[d(A-T)] and crab d(A-T) are effective templates. With crab d(A-T) or poly[d(A-T)] under template-limiting conditions synthesis is autocatalytic and continues until complete exhaustion of the input deoxyribonucleotides occurs. The size of the product after an S-fold net synthesis is approximately the same as that of the original template.