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DNA synthesis by the isolated nuclear matrix from synchronized plasmodia of Physarum polycephalum
M Shioda1, Y Matsuzawa, K Murakami-Murofushi
1Department of Physiological Chemistry and Nutrition, Faculty of Medicine, University of Tokyo, Japan.
Abstract:
Nuclear matrices were isolated from plasmodia of a true slime mold, Physarum polycephalum, and the DNA synthetic activity in vitro was examined. These matrices isolated in S-phase catalyzed DNA synthesis requiring Mg2+, deoxyribonucleoside 5'-triphosphates and ATP, without exogenous templates. The activity changed during S-phase with the rate of in vivo DNA replication. Product analysis by gel electrophoresis revealed that the matrices produced Okazaki fragments. These results suggest that DNA synthesis partially reflects in vivo DNA replication. DNA synthesis was sensitive to aphidicolin, heparin and N-ethylmaleimide, indicating involvement of the alpha-like DNA polymerase of Physarum. Exogenous addition of activated DNA stimulated DNA synthesis 4-10-fold and suggested that only some of the existing enzymes are involved in endogenous DNA synthesis. Matrices isolated in G2-phase were also associated with a similar DNA synthetic activity, but they did not produce Okazaki fragments in vitro. It is, therefore, concluded that nuclear matrices are associated with alpha-like DNA polymerase throughout the cell cycle, and that some of the enzymes participate in in vivo DNA replication in S-phase; thus, DNA replication is possibly controlled by this process. The relationship between DNA synthetic activities by the isolated nuclei and matrices was also discussed.
Insights
Nuclear matrices from Physarum polycephalum catalyze DNA synthesis, producing Okazaki fragments in S-phase. This suggests nuclear matrices play a role in controlling DNA replication via alpha-like DNA polymerase activity.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Nuclear matrices are structural components within the cell nucleus.
- Understanding the role of nuclear matrices in DNA replication is crucial for cell cycle regulation.
Purpose of the Study:
- To investigate the DNA synthetic activity of nuclear matrices isolated from Physarum polycephalum.
- To determine if nuclear matrices contribute to in vivo DNA replication processes.
Main Methods:
- Isolation of nuclear matrices from Physarum polycephalum plasmodia in S-phase and G2-phase.
- In vitro assay of DNA synthesis using isolated nuclear matrices.
- Analysis of synthesized DNA products, including Okazaki fragments, via gel electrophoresis.
- Assessment of DNA synthesis sensitivity to specific inhibitors (aphidicolin, heparin, N-ethylmaleimide).
Main Results:
- Nuclear matrices from S-phase Physarum polycephalum catalyzed template-independent DNA synthesis requiring Mg2+, deoxynucleoside triphosphates, and ATP.
- The observed DNA synthesis activity correlated with the in vivo replication rate and produced Okazaki fragments.
- DNA synthesis was inhibited by aphidicolin, heparin, and N-ethylmaleimide, implicating an alpha-like DNA polymerase.
- Nuclear matrices from G2-phase cells showed DNA synthetic activity but did not produce Okazaki fragments.
- Exogenous activated DNA stimulated endogenous synthesis, suggesting limited enzyme involvement in basal activity.
Conclusions:
- Nuclear matrices are associated with alpha-like DNA polymerase throughout the cell cycle in Physarum polycephalum.
- These matrices contribute to in vivo DNA replication during S-phase, potentially playing a role in its regulation.
- The findings highlight the functional significance of nuclear matrices in DNA replication processes.