Processivity of mitochondrial DNA polymerase from Drosophila embryos. Effects of reaction conditions and enzyme purity

• Published in: The Journal of biological chemistry 1993-11, Vol.268 (33), p.24855-24862
• Main Authors: Williams, A J, Wernette, C M, Kaguni, L S
• Format: Article
• Language: English
• Subjects: adenosine diphosphate; adenosine triphosphate; Adenosine Triphosphate - metabolism; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; DNA Polymerase III - isolation & purification; DNA Polymerase III - metabolism; DNA replication; DNA, Mitochondrial - biosynthesis; DNA, Single-Stranded - biosynthesis; DNA-directed DNA polymerase; Drosophila; Drosophila - enzymology; Drosophila melanogaster; embryo (animal); Enzyme Activation; enzyme activity; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; glycerol; Glycerol - pharmacology; guanosine triphosphate; Hydrogen-Ion Concentration; Larva - enzymology; Magnesium Chloride; Mitochondria - enzymology; mitochondrial DNA; polyethylene glycol; Potassium Chloride; salinity; Templates, Genetic; Transferases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)74543-8

Mitochondrial DNA polymerase from Drosophila embryos has been characterized with regard to its mechanism of DNA synthesis under the influence of a variety of compounds in moderate salt (120 mM KCl), where the enzyme is most highly active and only moderately processive, and in low salt (30 mM KCl), where it is less active yet most highly processive. Disparate activity and processivity optima were obtained in low salt in the presence of varying pH or MgCl2 and ATP concentrations; in moderate salt, optimal activity and processivity were achieved coincidentally. Whereas no correlation between processivity and activity optima was observed upon addition of polyethylene glycol in either low or moderate salt, the optima were coincident at both salt levels on addition of glycerol. None of the reaction conditions examined allowed DNA polymerase gamma to exhibit maximal activity and processivity concurrently; maximal activity was always achieved in moderate salt and the highest processivity in low salt. However, while limiting the availability of primer termini had no effect on the mechanism of DNA synthesis, we found that the ability of mitochondrial DNA polymerase to copy singly primed M13 DNA was enhanced then diminished during the course of purification, suggesting loss of an accessory factor.