Deoxyribonucleic acid polymerase from nuclei of rat intestinal mucosa

Abstract

An extract with DNA polymerase activity was prepared from nuclei of intestinal mucosa of the rat. Chromatography of the crude extract on DEAE-cellulose yielded three enzymically active fractions: I, II, and III. Each fraction could be resolved further into two components with DNA polymerase activity by rechromatography on smaller columns of DEAE-cellulose. A similar result was obtained by gel filtration of fraction II material on Sephadex G-150. The result of sucrose density gradient centrifugation of the fractions obtained by gel filtration suggested that each still consisted of a mixture of proteins with DNA polymerase activity. The approximate molecular weights of the active proteins, estimated by comparison with marker proteins, ranged from 25 000 to 300 000. Partially purified DNA polymerase (fraction II) required for activity the four deoxyribonucleoside triphosphates, Mg2+, 2-mercaptoethanol, and DNA template. The optimum pH for activity was 8.0 in Tris–HCl buffer and 7.4 in phosphate buffer. The two components obtained by gel filtration of fraction II differed in their requirements for DNA template. The one of smaller molecular size was more active with native DNA whereas the larger was equally active with either native or heat-denatured DNA.