DNA polymerase activity associated with purified nuclei of rat intestinal mucosa was studied. Two DNA polymerase activities have been isolated, partially purified and characterized.
One of the enzymes was extracted from purified nuclei with 10 mM Tris-HCl, pH 8.0, containing 5 mM dithiothreitol while the second enzyme, which was associated with the nuclear deoxyribonucleoprotein complex, was extracted only in a high ionic strength medium containing 1 M NaCl in 0.1 M Tris-HCl, pH 8.0 and 5 mM dithiothreitol. The molecular weights of these nuclear DNA polymerases were estimated by gel filtration on Sephadex G-150. Two peaks of DNA polymerase activity were detected when the Tris-soluble extract was chromatographed. The molecular weights of these peaks of activity were calculated to be 266,000 and 104,000. It was concluded that the first peak of activity represented an aggregate of the second. A single peak of DNA polymerase activity was obtained when the NaCl-soluble nuclear extract was chromatographed on Sephadex G-150. It corresponded to a molecular weight of approximately 40,000.
Chromatography on DEAE-cellulose indicated that the two enzymes differed in ionic charge. The bulk of the Tris-soluble DNA polymerase activity eluted with 0.045-0.055 M KCl, while the NaCl-soluble enzyme had a higher affinity for the anion exchange resin and was not eluted until the KCl concentration was 0.165-0.21 M. The partially purified enzymes were very labile. Storage at 4°C, 0°C or -20°C did not increase enzyme stability. The presence of glycerol, which had no effect on
enzyme activity, helped maintain the stability of both enzymes for at
least 1 month at -20°C.
The enzymic properties of the nuclear DNA polymerases differed.
In Tris-HCl buffer, a pH of 7.5 was optimal for the polymerase reaction
catalyzed by either enzyme, but in phosphate buffer the pH optima were
7.2 and 6.0 for the Tris-soluble and NaCl-soluble enzymes, respectively.
The presence of DNA, all 4 deoxynucleoside 5'-triphosphates and Mg²⁺
ions was required for the activity of both crude and partially purified
forms of the nuclear DNA polymerases. Substitution of Mn²⁺ or Ca²⁺
for Mg²⁺ resulted in lower enzymic activity. The addition of dithio-threitol greatly enhanced the activity of both enzymes, especially the purified preparations. The presence of thiol reagents, p-hydroxy-mercuribenzoate and N-ethylmaleimide, inhibited both of the nuclear DNA polymerase activities. In the presence of 1 mM nalidixic acid the activity of the Tris-soluble enzyme was abolished whereas the NaCl-soluble DNA polymerase activity was greatly enhanced. The activities of the nuclear DNA polymerases were also affected differently by monovalent cations. The addition of NH₄⁺, K⁺ or Na⁺ to the assay mixture inhibited the activity of the Tris-soluble enzyme but stimulated by 30-170% the activity of the NaCl-soluble DNA polymerase. The two enzymes also differed in template preference. The crude Tris-soluble DNA polymerase functioned equally well with either heat-denatured or native DNA, while the purified form showed a slight preference for native over heat-denatured DNA. The purified NaCl-soluble DNA polymerase, which in crude extracts consistently preferred native DNA as template, showed a strict dependence on native DNA.
Differences between the Tris-soluble and NaCl-soluble DNA polymerases in extractability from purified nuclei, molecular weight, ionic charge and in their enzymic properties clearly indicate that two distinct DNA polymerase activities are associated with purified nuclei prepared from rat intestinal mucosa cells. The close association between the NaCl-soluble DNA polymerase and the deoxyribonucleoprotein complex and its absolute dependence on native DNA template support the conclusion that it is a repair enzyme in vivo. The role of the Tris-soluble enzyme is less certain. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/32693 |
| Date | January 1973 |
| Creators | Krasny, Jiri Ladislav |
| Publisher | University of British Columbia |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
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