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Reconstitution of an ATM-dependent checkpoint that inhibits chromosomal DNA replication following DNA damage
V Costanzo1, K Robertson, C Y Ying
1Department of Genetics and Development, Columbia University, New York, NY 10032, USA.
Abstract:
Cell cycle checkpoints lead to the inhibition of cell cycle progression following DNA damage. A cell-free system derived from Xenopus eggs has been established that reconstitutes the checkpoint pathway inhibiting DNA replication initiation. DNA containing double-strand breaks inhibits replication initiation in a dose-dependent manner. Upon checkpoint activation, a prereplicative complex is assembled that contains ORC, Cdc6, Cdc7, and MCM proteins but lacks Cdc45. The checkpoint is ATM dependent. Cdk2/CyclinE acts downstream of ATM and is downregulated by Cdk2 phosphorylation on tyrosine 15. Cdk2AF/CyclinE is refractory to checkpoint signaling, and Cdc25A overrides the checkpoint and restores DNA replication. This report provides the description of a DNA damage checkpoint pathway that prevents the onset of S phase independently of the transcriptional function of p53 in a vertebrate organism.
Insights
This study reveals a DNA damage checkpoint pathway in Xenopus that halts DNA replication initiation. This ATM-dependent pathway prevents S phase entry, independent of p53, by regulating key replication proteins.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Cell cycle checkpoints prevent cell cycle progression after DNA damage.
- A Xenopus cell-free system reconstitutes the DNA replication checkpoint pathway.
Purpose of the Study:
- To describe a DNA damage checkpoint pathway that inhibits DNA replication initiation.
- To investigate the molecular mechanisms of this checkpoint in a vertebrate system.
Main Methods:
- Utilized a Xenopus egg-derived cell-free system.
- Assessed the dose-dependent inhibition of replication initiation by double-strand breaks.
- Analyzed protein complex assembly and phosphorylation events.
Main Results:
- DNA double-strand breaks inhibit replication initiation in a dose-dependent manner.
- Checkpoint activation involves ATM, Cdk2/CyclinE, and affects prereplicative complex formation.
- Cdc25A overrides the checkpoint, restoring DNA replication.
Conclusions:
- A novel DNA damage checkpoint pathway prevents S phase entry independently of p53 transcriptional activity.
- This pathway involves ATM-dependent regulation of Cdk2/CyclinE and prereplicative complex components.