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Cdc45 protein-single-stranded DNA interaction is important for stalling the helicase during replication stress
Irina Bruck1, Daniel L Kaplan1
1Florida State University College of Medicine, Department of Biomedical Sciences, Tallahassee, Florida 32306.
The Journal of Biological Chemistry
|February 6, 2013
Summary
Cdc45 binds single-stranded DNA, crucial for coordinating replication and helicase stalling during stress. A Cdc45 mutant lacking this interaction inhibits cell growth and uncouples DNA polymerase and helicase movement.
Area of Science:
- Molecular Biology
- DNA Replication
- Cell Cycle Regulation
Background:
- Replicative polymerase and helicase stalling are coordinated in eukaryotes.
- The mechanism coordinating this process remains unclear.
- Cdc45 is a known activator of the Mcm2-7 helicase.
Purpose of the Study:
- To investigate the role of Cdc45 in coordinating DNA replication and helicase activity.
- To elucidate the mechanism of helicase stalling during replication stress.
- To determine the importance of Cdc45-DNA interaction in this process.
Main Methods:
- Biochemical assays to study Cdc45-DNA binding.
- Genetic analysis of a Cdc45 mutant in budding yeast.
- Hydroxyurea treatment to induce replication stress.
- Chromatin immunoprecipitation to assess protein-DNA interactions and protein movement.
Main Results:
- Budding yeast Cdc45 binds tightly to long single-stranded DNA (ssDNA).
- ssDNA disrupts the Cdc45-Mcm2-7 helicase interaction.
- A Cdc45 mutant unable to bind ssDNA causes severe growth inhibition and RPA accumulation under replication stress.
- Chromatin immunoprecipitation indicates uncoupled helicase and polymerase movement in mutant cells.
Conclusions:
- The interaction between Cdc45 and ssDNA is essential for proper helicase stalling during replication stress.
- This interaction plays a key role in coordinating replicative polymerase and helicase activity.
- Cdc45-ssDNA binding is a critical component of the DNA replication checkpoint mechanism.
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