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DNA Polymerases I and II in Regenerating Rat Liver and Morris Hepatomas¹

Departments of Physiology and Pharmacology [E. F. B.] and Medicine [O. E. B., J. L.], Duke University Medical Center and Veterans Administration Hospital [M. D. J., J. L.], Duke University, Durham, North Carolina 27710, and Department of Biochemistry, College of Medicine, Howard University, Washington, D. C. 20001 [H. P. M.]

Two nonmitochondrial forms of DNA polymerase are demonstrated in regenerating rat liver and Morris hepatomas 7777, 7800, and 7794A. The enzymes have been partially purified from subcellular fractions of these tissues. The purification procedure involves fractionation with ammonium sulfate and chromatography on diethylaminoethyl cellulose and on phosphocellulose. The enzymes have different physical and enzymatic properties and have been designated DNA polymerases I and II. The properties of the respective DNA polymerases from normal and regenerating rat liver and the Morris hepatomas appear to be identical. The purified enzymes show a strict requirement for a polydeoxyribonucleotide template and primer. Double-stranded DNA into which 3'-hydroxyl termini have been introduced by pancreatic DNase is the preferred primer for the purified DNA polymerases. Both enzymes require all four deoxyribonucleoside triphosphates for maximal activity. DNA polymerases I and II exhibit maximal activity at 9.0 and 8.0 pH units and at 15 and 10 mM Mg²⁺, respectively. The activity of DNA polymerase II is completely inhibited by thiol-blocking agents at a concentration of 0.6 mM, while the activity of DNA polymerase I is not inhibited. The polymerases have different net charges as shown by their distinctive affinities for diethylaminoethyl cellulose and phosphocellulose. The molecular weights of DNA polymerases I and II were estimated by gel filtration as 25,000 and 250,000, respectively.

DNA polymerase I is present in the nuclei and with cytoplasmic ribosomes from normal and regenerating rat liver and hepatomas. This is the predominant DNa polymerase activity in normal rat liver and the activity is not increased significantly in regenerating rat liver or in hepatomas. In contrast, the activity of DNA polymerase II is low in normal rat liver and is detectable only with a smooth membrane fraction that is isolated from the postmicrosomal supernatant solution. In regenerating rat liver and hepatomas DNA polymerase II is present in the nucleus, as well as the smooth membrane fraction. The activity of DNA polymerase II with these subcellular fractions increases between 18 and 30 hr posthepatectomy and then declines. A substantial increase (six- to tenfold) in the activity of DNA polymerase II relative to normal rat liver was observed in the nuclear and smooth membrane fractions from the Morris hepatomas.

¹ This investigation was supported by USPHS Research Grants Ca-08800-04, Ca-11265, and Ca-10729 and the Duke Endowment Fund.

² Present address: The Worcester Foundation for Experimental Biology, Shrewsbury, Mass. 01545.

Received 6/20/72. Accepted 3/ 1/73.