Characterization and properties of a DNA polymerase partially purified from the nuclei of calf thymus cells

Abstract

The novel DNA polymerase recently isolated from the nuclei of calf thymocytes in our laboratory was characterized and its distinctive properties were determined. The enzyme worked most efficiently at pH near 7.0 and catalyzed a replicative DNA polymerization on suitable primer templates using Mg 2+ or Mn 2+ as a metal cofactor and the complementary deoxynucleoside triphosphates as substrates. The active protein was inhibited by 0.1 M KCl, combined steadily with DNA—cellulose and behaved in gel filtration as a globulin with an approximate molecular weight of 73 000. When expressed in terms of total nucleotide polymerized under optimal conditions, the rates of the enzymatic reactions directed respectively by native, denatured and “activated” DNA and by (dA) n · (dT) 12 as primers were quantitatively related according to the numerical proportion 1.0:1.2:3.0:5.1. Devoid of any detectable deoxyribonuclease contamination, the partially purified preparation carried out a simple duplication of single-stranded DNA and a several-fold replication of bihelical DNA, both the DNA products being probably linked to the templates by covalent bonds. The other known DNA polymerases from calf thymus were also partially purified and used to probe some of the specific properties which differentiated them from the newly isolated enzyme. For instance, the nuclear 3.39 S deoxynucleotidyltransferase exhibited comparatively: lower molecular weight, more alkaline pH optimum, other activity ratios with the various primers, relatively more preference for initiated templates such as “activated” DNA and (dA) n · (dT) 12, and much more ability to function on templates of the ribose series; moreover, it may be expected to undergo stimulation instead of inhibition at 0.1 M salt concentrations. On the other hand, the soluble 6–8 S DNA polymerase had a higher molecular weight and diverse subcellular location, and it may be expected not to complex with DNA—cellulose.