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DNA replication in Physarum polycephalum: electron microscopic analysis of patterns of DNA replication in the
Abstract:
DNA from synchronously replicating nuclei of Physarum polycephalum was studied electron microscopically after 15, 30, 60, and 90 or 120 min of replication in the presence or absence of the protein synthesis inhibitor cycloheximide. The replication-loop size-distribution showed that replication fork progression is severely retarded in the presence of cycloheximide. Analysis of replication-loop frequency showed a similar pattern in control and cyclo-heximide-treated samples, with an increase from 15 to 30 and 60 min. This suggests, surprisingly, that initiations of new replicons either may not be inhibited by cycloheximide or, alternatively, that all initiations have already taken place at the very start of S-phase. The latter conclusion is favored in the light of previous results in our laboratory, discussed here.
Insights
Protein synthesis inhibition by cycloheximide significantly slows DNA replication fork progression in Physarum polycephalum. However, new replicon initiations appear unaffected, suggesting they occur early in the S-phase.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Understanding DNA replication is crucial for cell division and organism development.
- Protein synthesis plays a role in regulating DNA replication processes.
- Physarum polycephalum serves as a model organism for studying nuclear processes.
Purpose of the Study:
- To investigate the effect of cycloheximide, a protein synthesis inhibitor, on DNA replication in Physarum polycephalum.
- To analyze replication fork progression and replicon initiation under inhibited protein synthesis conditions.
- To elucidate the role of protein synthesis in the regulation of DNA replication initiation.
Main Methods:
- Electron microscopy was used to examine DNA from synchronously replicating Physarum polycephalum nuclei.
- Samples were collected at various time points (15, 30, 60, 90-120 min) during replication.
- Experiments were conducted both in the presence and absence of the protein synthesis inhibitor cycloheximide.
Main Results:
- Cycloheximide treatment severely retarded replication fork progression, as indicated by replication-loop size distribution.
- Replication-loop frequency showed a similar increasing pattern in both control and cycloheximide-treated samples over time.
- These findings suggest that new replicon initiations were not inhibited by cycloheximide or occurred prior to treatment.
Conclusions:
- Protein synthesis inhibition by cycloheximide significantly impacts DNA replication fork speed.
- Replicon initiation in Physarum polycephalum appears to be independent of ongoing protein synthesis during S-phase.
- The data favor the conclusion that replicon initiations occur early in S-phase, before significant protein synthesis is required.