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An ATR- and Cdc7-dependent DNA damage checkpoint that inhibits initiation of DNA replication
Vincenzo Costanzo1, David Shechter, Patrick J Lupardus
1Department of Genetics and Development, Columbia University, New York, NY 10032, USA.
Abstract:
We have analyzed how single-strand DNA gaps affect DNA replication in Xenopus egg extracts. DNA lesions generated by etoposide, a DNA topoisomerase II inhibitor, or by exonuclease treatment activate a DNA damage checkpoint that blocks initiation of plasmid and chromosomal DNA replication. The checkpoint is abrogated by caffeine and requires ATR, but not ATM, protein kinase. The block to DNA synthesis is due to inhibition of Cdc7/Dbf4 protein kinase activity and the subsequent failure of Cdc45 to bind to chromatin. The checkpoint does not require pre-RC assembly but requires loading of the single-strand binding protein, RPA, on chromatin. This is the biochemical demonstration of a DNA damage checkpoint that targets Cdc7/Dbf4 protein kinase.
Insights
Single-strand DNA gaps trigger a DNA damage checkpoint in Xenopus egg extracts, halting DNA replication by inhibiting Cdc7/Dbf4 kinase. This ATR-dependent checkpoint requires RPA loading but not pre-RC assembly.
Area of Science:
- Molecular Biology
- Cellular Biology
- Biochemistry
Background:
- DNA replication is a fundamental process susceptible to damage.
- DNA damage checkpoints are crucial for maintaining genomic integrity.
- Understanding replication checkpoints informs cancer research and drug development.
Purpose of the Study:
- To investigate the role of single-strand DNA gaps in activating DNA damage checkpoints.
- To elucidate the molecular mechanisms by which DNA damage inhibits DNA replication initiation.
- To identify key proteins and pathways involved in this checkpoint response.
Main Methods:
- Utilized Xenopus egg extracts for in vitro DNA replication studies.
- Induced DNA lesions using etoposide (DNA topoisomerase II inhibitor) and exonuclease treatment.
- Assessed checkpoint activation by monitoring DNA replication initiation and protein kinase activity.
- Investigated protein requirements (ATR, ATM, RPA, Cdc45) and caffeine sensitivity.
Main Results:
- Single-strand DNA gaps activate a DNA damage checkpoint, blocking replication initiation.
- The checkpoint is dependent on ATR protein kinase and abrogated by caffeine.
- Replication inhibition results from Cdc7/Dbf4 protein kinase inactivation and impaired Cdc45 chromatin binding.
- Checkpoint activation requires RPA loading onto chromatin but not pre-replicative complex assembly.
Conclusions:
- This study provides the first biochemical evidence of a DNA damage checkpoint targeting the Cdc7/Dbf4 protein kinase.
- Single-strand DNA gaps serve as a signal for this replication-blocking checkpoint.
- The findings reveal a novel mechanism for controlling DNA replication in response to DNA damage.