Sub-cellular localization and characterization of DNA polymerases from rat liver and hepatomas

Abstract

Summary Previous studies from this laboratory described a correlation between DNA synthesis and DNA polymerase activity in a variety of normal rat tissues, regenerating rat liver and a series of 12 types of rat hepatomas. DNA polymerase in these tissues, isolated by the Mantsavinos procedure, was separated into two peaks of activity by Sephadex G-200 gel filtration. A low molecular weight fraction prefers native DNA primer, is the predominant component of normal rat liver and increases markedly in regenerating liver. A fraction of high molecular weight material which prefers denatured DNA as primer is present to a small extent in normal and regenerating rat liver but increases markedly in fetal rat liver (−6 days from birth), other developing tissues of the rat, rat hepatomas, rat renal tumors and various human cancers. Sub-cellular fractionation of rat liver and selected Morris hepatomas showed that DNA polymerase activity preferring denatured DNA primer is associated with a cytoplasmic smooth membrane fraction in normal liver and is present in the nucleus, as well as with the smooth membranes from hepatoma tissues. The activity with these fractions from the progressed hepatoma 7777 was markedly increased. DNA polymerase preferring native DNA as primer is associated with the nucleus and ribosomes of rat liver and hepatoma. DNA polymerase associated with these sub-cellular fractions from rat liver and hepatoma tissues was partially purified and the properties of the polymerase compared. The ribosomal- and nuclear-associated polymerase of rat liver appears to be the same enzyme in that the physical and enzymatic properties of the purified enzyme are identical. The membrane-associated DNA polymerase, also present in nuclei of hepatomas, has different physical and enzymatic properties than the polymerase associated with both ribosomes and nuclei of liver. After purification both enzymes prefer “activated DNA” as primer. Purified membrane polymerase is free of nuclease activity but purified ribosome polymerase contains a small amount of endonuclease activity that produces single-strand breaks in DNA (“nickase”). All four deoxyribonucleoside triphosphates are required for normal activity of both polymerases but the purified polymerase from ribosomes has about 25% maximal activity in the presence of a single deoxyribonucleoside triphosphate. This activity does not appear to catalyze terminal addition to the primer since experiments with homodeoxyribopolymers demonstrated a requirement for base pairing with the template. We suggest that the physiological function of the polymerase associated with ribosomes and nuclei is in limited polymerization reactions such as DNA repair. The polymerase associated with smooth membranes, and present in the nuclei of proliferating tissues, may function in the replication reaction. Furthermore, we support earlier suggestions ( 18. , 33. ) that the presence of these enzymes in cytoplasm may be due to their origin in this compartment of the cell.