DNA-Dependent DNA Polymerase from Yeast Mitochondria

Abstract

The activity of DNA polymerase was determined in gradient-purified mitochondria from yeast cells grown under a variety of conditions. The specific enzyme activity was found to be dependent on the degree of aeration of the cells, and on the carbon source used for the medium. It was sensitive to glucose repression, and was enhanced about two-fold by the growth of yeast cells in the presence of ethidium bromide.

Mitochondrial DNA polymerase was highly purified and several properties were determined. Sucrose density gradient centrifugation, and dodecylsulfate-polyacrylamide gel electrophoresis revealed the following structure: a monomer of molecular weight around 60000 aggregated under relatively high salt concentration (0.2 M phosphate buffer) to a dimer of about 120000 which under low salt concentration (0.02 M Tris-HCI buffer) formed higher aggregates. For optimal activity an Mg²⁺ ion concentration of 50 mM was found necessary, Mn ions did not promote activity at any concentration tested (0.5-50 mM). Indeed, if added to Mg²⁺ containing assays, Mn²⁺ strongly inhibited enzyme activity at low concentrations. This might be an explanation for the inducation of mitochondrial mutants in yeast cells grown in the presence of Mn²⁺ ions. Mitochondrial DNA polymerase activity was strongly inhibited by low concentrations of the -SH reagent p-chloromercuribenzoate, the nucleotide analogue cytosine arabinoside triphosphate also exerted an inhibitory effect. An about 50% decrease of activity was observed in the presence of 1 mM o-phenanthroline in assay mixture containing DNA at about the K_m concentration. The enzyme preferred a gapped template primer, poly(dA) · (dT)₁₀, over nicked DNA and was unable to use a polyrihonucleotide template, poly(rA) · (dT)₁₀. In the purest preparations no exonuclease activity could he detected.