Purification and Properties of Deoxyribonucleic Acid Polymerase from Rat Liver Mitochondria

Abstract

Abstract These studies report the isolation of a DNA polymerase from rat liver mitochondria. The enzyme has been purified 22-fold by differential ultracentrifugation, ammonium sulfate precipitation, and column chromatography on DEAE-cellulose. The partially purified enzyme manifests all of the requirements shown by the known DNA polymerases of mammalian nuclei and bacteria. The enzyme has an optimal pH of 7.5, and requires Mg++ (12 mm) and the presence of all four deoxyribonucleoside triphosphates. The reaction is inhibited by pyrophosphate and by the addition of deoxyribonuclease but not by ribonuclease. Enzyme purified in this manner is free of nuclear DNA polymerase, terminal addition enzyme, and DNase. The enzyme shows an absolute requirement for added DNA template which can be furnished rather specifically by native, double-stranded, circular, mitochondrial DNA. Furthermore, the labeled DNA was shown to be mitochondrial in nature by virtue of the fact that it had the same buoyant density in CsCl as unlabeled, highly purified, mitochondrial DNA and the fact that it was renaturable after heat denaturation, a property shown by mitochondrial DNA but not by mammalian nuclear DNA. The product of the reaction was shown to be a double stranded replica of the mitochondrial DNA template on the basis of autocatalytic synthesis by the enzyme with the use of nonsaturating levels of mitochondrial DNA template. A 3.5-fold synthesis of new DNA was achieved.