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DNA synthesis in isolated nuclei from synchronized plasmodia of Physarum polycephalum
Abstract:
Nuclei were isolated from synchronized plasmodia of a true slime mold, Physarum polycephalum, in S-phase, and DNA synthesis in the nuclei was studied in vitro. The nuclei catalyzed DNA synthesis at the rate of 0.7 ng DNA/1.0 X 10(6) nuclei/30 min at 25 degrees C, which was 5 times higher than that catalyzed in G2-phase nuclei. The DNA synthesis required Mg2+, four kinds of deoxyribonucleoside 5'-triphosphates and ATP, suggesting that the mode of synthesis is a replicative-type, but not a repair-one. Sedimentation analysis of the DNA products revealed that the nuclei produced 2-4S DNA fragments mainly during a 30-sec pulse incubation, and 2-4S, 5-12S and longer fragments during a 15-min incubation. The pulse- and chase-labeling experiments showed that the 2-4S fragments shifted discontinuously to longer fragments. These results indicate that the nuclei catalyze the formation of 2-4S Okazaki fragments first and then their subsequent ligation. Eighty % and 96% of the DNA synthesis was inhibited by 200 micrograms/ml aphidicolin and 40 mM N-ethylmaleimide, respectively, but 80% of the activity was resistant to 100 microM 2',3'-dideoxythymidine 5'-triphosphate. These results suggest that the DNA synthesis is catalyzed by the alpha-type DNA polymerase of Physarum polycephalum.
Insights
Physarum polycephalum nuclei synthesize DNA via a replicative mechanism, forming Okazaki fragments and ligating them. This process is primarily driven by alpha-type DNA polymerase.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Understanding DNA replication mechanisms is crucial for cell division and genetic stability.
- Physarum polycephalum, a true slime mold, offers a unique model for studying nuclear DNA synthesis.
Purpose of the Study:
- To investigate the in vitro DNA synthesis activity of Physarum polycephalum nuclei during the S-phase.
- To characterize the type of DNA synthesis and identify the key enzymes involved.
Main Methods:
- Isolation of synchronized S-phase nuclei from Physarum polycephalum plasmodia.
- In vitro assay of DNA synthesis rates and product analysis using sedimentation.
- Inhibition studies with aphidicolin, N-ethylmaleimide, and 2',3'-dideoxythymidine 5'-triphosphate.
Main Results:
- S-phase nuclei exhibited significantly higher DNA synthesis rates compared to G2-phase nuclei.
- DNA synthesis produced 2-4S Okazaki fragments, which were subsequently elongated and ligated.
- Aphidicolin and N-ethylmaleimide strongly inhibited DNA synthesis, while 2',3'-dideoxythymidine 5'-triphosphate showed resistance.
Conclusions:
- Physarum polycephalum nuclei perform replicative DNA synthesis, involving Okazaki fragment formation and ligation.
- The results strongly suggest the involvement of an alpha-type DNA polymerase in this process.