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In vitro cell cycle arrest induced by using artificial DNA templates
S Kornbluth1, C Smythe, J W Newport
1Department of Biology, University of California, San Diego, La Jolla 92093.
Abstract:
In cell extracts of Xenopus eggs which oscillate between S and M phases of the cell cycle, the onset of mitosis is blocked by the presence of incompletely replicated DNA. In this report, we show that several artificial DNA templates (M13 single-stranded DNA and double-stranded plasmid DNA) can trigger this feedback pathway, which inhibits mitosis. Single-stranded M13 DNA is much more effective than double-stranded plasmid DNA at inhibiting the onset of mitosis. Furthermore, we have shown that low levels of M13 single-stranded DNA and high levels of double-stranded plasmid DNA can elevate the tyrosine kinase activity responsible for phosphorylating p34cdc2, thereby inactivating maturation-promoting factor and inhibiting entry into mitosis. This constitutes a simplified system with which to study the signal transduction pathway from the DNA template to the tyrosine kinase responsible for inhibiting p34cdc2 activity.
Insights
Artificial DNA templates like M13 single-stranded DNA can block mitosis in Xenopus egg extracts by triggering a feedback pathway. Single-stranded DNA is more effective than double-stranded DNA at inhibiting cell cycle progression.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Cell cycle progression is regulated by checkpoints that prevent entry into mitosis until DNA replication is complete.
- In Xenopus egg extracts, incompletely replicated DNA triggers a feedback mechanism that inhibits the onset of mitosis.
Purpose of the Study:
- To investigate the role of artificial DNA templates in triggering the DNA replication checkpoint.
- To elucidate the signal transduction pathway from DNA templates to the inhibition of mitosis.
Main Methods:
- Utilized Xenopus egg extracts as a cell-free system.
- Introduced artificial DNA templates, including M13 single-stranded DNA and double-stranded plasmid DNA.
- Measured tyrosine kinase activity and p34cdc2 phosphorylation levels.
Main Results:
- Both single-stranded M13 DNA and double-stranded plasmid DNA inhibited mitosis in Xenopus egg extracts.
- Single-stranded M13 DNA was more potent than double-stranded plasmid DNA in blocking mitosis.
- Elevated tyrosine kinase activity, leading to p34cdc2 phosphorylation and maturation-promoting factor inactivation, was observed with specific DNA template concentrations.
Conclusions:
- Artificial DNA templates can effectively mimic the effects of incompletely replicated DNA in triggering the cell cycle checkpoint.
- The study provides a simplified system to analyze the DNA-to-tyrosine kinase signal transduction pathway regulating mitosis.
- Findings highlight the differential efficacy of single-stranded versus double-stranded DNA in activating this cell cycle inhibitory mechanism.