The Deoxyribonucleic Acid Nucleotidyltransferase Activities of the Shope Fibroma: Partial Purification and Kinetics

Abstract

Abstract DNA nucleotidyltransferase was partially purified from Shope fibroma tissue. The final preparation was free of terminal transferase but contained traces of deoxyribonuclease and nonspecific phosphodiesterase. The preparation was active with either native or denatured DNA as template. When assayed with native DNA as template, the activity was purified 61-fold, whereas when denatured DNA was used as template, purification was 179-fold. The Km for deoxyribonucleoside triphosphates for the native DNA- and denatured DNA-primed activities were 0.046 mm and 0.16 mm, respectively. The Km for native DNA varied with the source of the DNA. The denatured DNA reaction alone was inhibited by high concentrations of substrate. The Km values (20 to 25 µg per ml) for denatured DNA were almost the same for denatured DNA from four different sources, but the K's values varied widely (90 to 650 µg per ml). Brief treatment with DNase I greatly increased the Vmax of the native but not the heat-denatured DNA-primed reaction. The Km of the native DNA-primed reaction was not affected. Despite the affinities for template demonstrable kinetically, the DNA nucleotidyltransferases and DNA failed to form complexes that were stable in neutral sucrose gradients. The data for the reaction primed by denatured DNA suggest competition by 2 molecules of denatured DNA for a secondary binding site for template DNA. The increase in Vmax but not in Km of the reaction primed by native DNA after partial digestion of the template by DNase I may have been due to the increased ease of unwinding of the molecule after the introduction of single strand breaks, whereas the failure of the same preparations to exhibit enhanced priming activity after denaturation indicates the necessity for DNA of some minimum length for the denatured DNA-primed enzyme activity. The data suggest that the preparation contains two different DNA nucleotidyl-transferases, but the possibility that a single molecule is responsible for both activities is not ruled out.