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Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence
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Oncogenes induce genotoxic stress by mitotic processing of unusual replication intermediates
Kai J Neelsen1, Isabella M Y Zanini, Raquel Herrador
1Institute of Molecular Cancer Research, University of Zurich, CH-8057 Zurich, Switzerland.
The Journal of Cell Biology
|March 13, 2013
Summary
Oncogene activation causes replication stress, but DNA damage and response activation require cell cycle arrest or checkpoint defects. Nuclease activity is key to oncogene-induced genotoxicity.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Oncogene-induced DNA replication stress activates the DNA damage response (DDR), a critical anticancer mechanism.
- Inactivation of DDR promotes genome instability and tumor progression, but the molecular basis is unclear.
Purpose of the Study:
- To elucidate the molecular mechanisms underlying oncogene-induced genotoxicity and DDR activation.
- To investigate the role of replication fork dynamics and nucleases in response to oncogene overexpression.
Main Methods:
- Overexpression of Cyclin E and Cdc25A to induce replication stress.
- Analysis of replication fork speed, fork reversal, chromosomal breakage, and DDR activation.
- Depletion of the nuclease MUS81 to assess its role.
Main Results:
- Overexpression of Cyclin E and Cdc25A slowed replication forks and induced reversal, without immediate DNA breakage or significant DDR activation.
- DNA breakage and DDR activation occurred upon persistent G2/M arrest or premature CDK1 activation in checkpoint-defective cells.
- MUS81 depletion reduced chromosomal breakage and increased reversed forks.
Conclusions:
- Nucleolytic processing of aberrant replication intermediates drives oncogene-induced genotoxicity.
- DNA damage checkpoints limit this nucleolytic processing to mitosis, acting as an anti-tumorigenic mechanism.
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