DNA-Dependent DNA Polymerase from Yeast Mitochondria. Dependence of Enzyme Activity on conditions of Cell Growth, and Properties of the Highly Purified Polymerase

Abstract

The activity of DNA polymerase [EC 2.7.7.7] was determined in gradient-purified mitochondria from yeast [Saccharomyces cerevisiae] 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 C source used for the medium. It was sensitive to glucose repression, and was enhanced about 2-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 MW around 60,000 aggregated under relatively high salt concentration (0.2 M phosphate buffer) to a dimer of about 120,000 which under low salt concentration (0.02 M Tris-HCl 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 may be an explanation for the indication 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 analog cytosine arabinoside triphosphate also exerted an inhibitory effect. Approximately 50% decrease of activity was observed in the presence of 1 mM o-phenanthroline in assay mixture containing DNA at about the Km concentration. The enzyme preferred a gapped template primer, poly(dA) .cntdot. (dT)10, over nicked DNA and was unable to use a polyribonucleotide template, poly(rA) .cntdot. (dT)10. In the purest preparations no exonuclease activity could be detected.