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Separable DNA Polymerase Activities in Host Liver and Morris Hepatomas¹

Department of Anatomy and Cell Biology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15213 [P. O., M. L. C.], and Howard University Medical Center, Washington, D. C. 20001 [H. P. M.]

DNA polymerase activities were extracted from the cytoplasm and from nuclei of host liver and Morris hepatoma 7800 and 7777. An improved method of isolating hepatoma nuclei in high yield and in relatively pure form is reported. Three major activities were extracted, one from the cytoplasm and two from the nuclei. The cytoplasmic extract derived from hepatoma 7777 has a higher activity than cytoplasmic extract from hepatoma 7800, and cytoplasmic host liver extract has the lowest activity. Extraction of nuclei with potassium phosphate yields a polymerase activity that is present in about equal amounts in the three tissues. A second extraction of nuclei with NaCl yields a different kind of activity. This activity is increased in the hepatomas similar to the cytoplasmic activity. The three activities differ in their elution from diethylaminoethyl cellulose, their primer preference, their reaction to the presence of KCl in the incubation medium, their ability to incorporate label in the absence of the three unlabeled deoxyribonucleoside triphosphates, and their ability to form and acid-precipitable product with synthetic polymers as primer. The activity extracted from the nuclei with NaCl differed further from the other two activities in its pH optimum, Mg²⁺ concentration requirement, sedimentation on sucrose gradients, and Sephadex G-200 filtration pattern. A fourth activity was resolved in relatively low amounts on diethylaminoethyl cellulose chromatography. This activity appeared unstable and was not further analyzed.

Nuclei, after extraction with potassium phosphate under mild conditions, are mostly intact nuclei and incorporated label as well as unextracted nuclei in an in vitro system using whole nuclei as the only source of enzyme and primer. Unextracted nuclei were able to incorporate substantially more label if exogenous DNA primer was added to the system. KP₁-extracted nuclei could not respond to exogenous primer. The KP₁-extractable activity seems to be responsible for this incorporation with the exogenous DNA as primer observed with unextracted nuclei. The NaCl-extractable activity seems to be the activity incorporating label into the nuclear DNA. Hepatoma nuclei incorporated more label than did host liver nuclei in the system using either extracted or unextracted whole nuclei.

¹ This work was supported by USPHS Grant 5 RO1, CA 11637-03 PHRB.

Received 11/22/72. Accepted 3/ 7/73.