Deoxyribonucleic Acid Biosynthesis in Mitochondria

Deoxyribonucleic acid polymerase has been purified 3550-fold from rat liver mitochondria, which computes to 14,000-fold based on whole liver as the starting material. The enzyme is essentially free of deoxyribonuclease, phosphomonoesterase, phosphodiesterase, deoxyguanosine triphosphatase, ribonuclease, nuclear DNA polymerase, and terminal nucleotidyltransferase activities. It is stable when stored at -125° or colder, but loses activity rapidly at higher temperatures and during incubation. All four deoxynucleoside triphosphates are required for activity, the pH optimum of the polymerization reaction is 8.0 to 9.0 in Tris buffer, and there is an absolute dependence on a divalent cation. Mn2+ partially substitutes for Mg2+, while Co2+ and Ca2+ are ineffective. Unlike most DNA polymerases, the enzyme is greatly stimulated by high ionic strength. The optimal salt concentration and degree of stimulation varies with the ion used; maximal activity is obtained at 0.15 m NaCl or KCl. The polymerase seems unable to synthesize polydeoxy-adenylate-thymidylate copolymer in the absence of primer, and cannot incorporate a ribonucleoside triphosphate into DNA in the presence of either Mg2+ or Mn2+. The enzyme possesses an absolute requirement for DNA which can be met by preparations from a variety of sources. The purified enzyme shows a preference for single stranded DNA; the degree of preference varies among DNA preparations and is markedly influenced by DNA concentration, salt concentration, and nuclease activity.