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Purification and characterization of DNA polymerase alpha from plasmodia of Physarum polycephalum
1Institut für Biophysik und Physikalische Biochemie der Universität, Regensburg, Federal Republic of Germany.
Abstract:
DNA polymerase alpha from Physarum polycephalum has been purified from freshly harvested microplasmodia. An inhibitory activity was removed by precipitation with poly(ethyleneimine) and interfering type-beta-like DNA polymerase by chromatography on phosphocellulose. The preparation was free of endonucleases and exonucleases. The DNA-polymerizing polypeptide had a molecular mass of 140 kDa by polyacrylamide gel electrophoresis under denaturing conditions. It was contained in purified samples and in crude cell extracts. Peptides of smaller size that reacted with antibodies against this protein were generated during purification and prolonged standing. Molecular sizing under non-denaturing conditions resulted in high-molecular-mass forms. The type of isolated DNA polymerase was established on the basis of inhibition and template-primer utilization experiments underlying the classification of DNA polymerases from higher eucaryotes. The majority of the DNA-polymerizing activity was contained in the cell nucleus fraction and was inhibited by aphidicolin. The isoelectric point (pI) was 6.7 +/- 0.2, the pH optimum at pH 6.8, and the temperature optimum at 40 degrees C. Monovalent salts, Li+, Na+, NH+4, K+, were inhibitory except for small activation maxima at 10 mM, 75 mM and 100 mM in the case of Na+, NH+4 and K+ respectively. The bivalent cations Mg2+ and Mn2+ had broad activity maxima at 3-20 mM concentrations, which were shifted to 0.05-0.1 mM in the case of Mn2+ and synthetic DNA homopolymers. The numbers of molecules of DNA polymerases in Physarum nuclei were calculated and compared with the established number of replicons in plasmodia and with the number of molecules of DNA polymerases in higher eucaryotes.
Insights
Physarum polycephalum DNA polymerase alpha was purified and characterized. This DNA polymerase is aphidicolin-sensitive, indicating it
Area of Science:
- Molecular Biology
- Biochemistry
- Eukaryotic DNA Replication
Background:
- DNA polymerases are essential enzymes for DNA replication and repair in all organisms.
- Understanding the characteristics of DNA polymerase alpha is crucial for comprehending eukaryotic DNA replication mechanisms.
- Physarum polycephalum, a unique acellular slime mold, offers a model system for studying fundamental cellular processes.
Purpose of the Study:
- To purify and characterize DNA polymerase alpha from Physarum polycephalum.
- To determine the enzymatic properties and classification of this DNA polymerase.
- To compare its molecular and catalytic features with DNA polymerases from other eukaryotes.
Main Methods:
- Purification of DNA polymerase alpha using poly(ethyleneimine) precipitation and phosphocellulose chromatography.
- Enzyme characterization including molecular mass determination (SDS-PAGE), isoelectric point, pH and temperature optima, and cation requirements.
- Inhibition studies using aphidicolin and template-primer utilization assays to classify the polymerase type.
Main Results:
- A 140 kDa DNA-polymerizing polypeptide, identified as DNA polymerase alpha, was purified and found to be largely nuclear.
- The enzyme exhibited characteristics consistent with eukaryotic DNA polymerase alpha, including aphidicolin sensitivity.
- Optimal activity was observed at pH 6.8 and 40°C, with specific requirements for divalent cations (Mg2+, Mn2+) and salt concentrations.
Conclusions:
- DNA polymerase alpha from Physarum polycephalum shares significant similarities with its counterparts in higher eukaryotes.
- The purification and characterization provide insights into the replication machinery of this primitive eukaryote.
- Further studies can elucidate the role of this enzyme in the unique life cycle and genome dynamics of Physarum polycephalum.