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Partial Characterization of the DNA-dependent DNA Polymerases of Rat Liver and Hepatoma¹

Department of Biochemistry, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 [G. R. H., G. F. K.], and Department of Biochemistry, Howard University College of Medicine, Washington, D. C. 20001 [H. P. M.]

Four DNA-dependent DNA polymerases, which include a soluble nuclear polymerase, the mitochondrial polymerase, and two polymerases associated with the smooth membrane fraction, have been partially purified from normal rat liver and a fast-growing Morris hepatoma, 7777, by previously published procedures. The membrane-associated enzymes have been designated as the 0.10 and 0.25 smooth membrane polymerases on the basis of their elution from a diethylaminoethyl cellulose column with the respective concentration of KCl.

The polymerases were shown to be separate enzymes by their ability to use synthetic polymers as templates and their response to several inhibitors. The mitochondrial DNA polymerase was readily distinguished from the smooth membrane polymerases by its ability efficiently to utilize poly(dA)d(pT)₁₀ as template and by its complete inhibition by concentrations of ethidium bromide which do not inhibit the other enzymes. Furthermore, the mitochondrial enzyme is the only one of the four polymerases which is insensitive to inhibition by p-hydroxymercuribenzoate.

The various polymerases were also distinguished by the effects of monovalent salts and the alkaloid antibiotic, camptothecin, on their activities. NaCl (0.05 to 0.10 M) stimulates the mitochondrial and 0.10 smooth membrane polymerases but is inhibitory to the nuclear and 0.25 smooth membrane enzymes. Camptothecin inhibits the nuclear and 0.25 smooth membrane polymerases, stimulates the 0.10 smooth membrane enzyme, and has no effect on the mitochondrial polymerase.

¹ This research was supported in part by Grants DRG-1086 from the Damon Runyon Memorial Fund, CA 12714-01 to G. F. K., and GRS-RR 5414 and CA10729 to Howard University from the NIH.

² Special Fellow of the Leukemia Society of America.

³ To whom correspondence should be addressed.

Received 11/14/72. Accepted 1/30/73.