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A general protocol for evaluating the specific effects of DNA replication inhibitors
1University of Virginia School of Medicine, Department of Biochemistry, Charlottesville 22908.
Nucleic Acids Research
|August 25, 1993
Summary
Replication inhibitors hydroxyurea, aphidicolin, and cytosine arabinoside are ineffective. Mimosine and ciclopyrox olamine (CPX) inhibit DNA replication initiation, with CPX also effective in yeast for identifying target genes.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- DNA replication inhibitors are crucial for cancer chemotherapy and laboratory cell synchronization.
- Understanding the precise mechanisms of these inhibitors is essential for optimizing their use.
Purpose of the Study:
- To investigate the specific effects of five different DNA replication inhibitors on cultured mammalian cells.
- To differentiate between inhibitors that block chain elongation versus those that inhibit initiation.
Main Methods:
- Utilized isotopic labeling protocols combined with two-dimensional gel replicon mapping.
- Assessed the impact of hydroxyurea, aphidicolin, cytosine arabinoside, mimosine, and ciclopyrox olamine (CPX) on DNA replication fork progression and initiation.
Main Results:
- Hydroxyurea, aphidicolin, and cytosine arabinoside showed limited effectiveness in preventing fork progression.
- Mimosine and ciclopyrox olamine (CPX) demonstrated inhibition of DNA replication initiation at origins.
- CPX also inhibited replication in yeast, suggesting a conserved target.
Conclusions:
- CPX and mimosine represent a distinct class of DNA replication inhibitors targeting initiation.
- The effectiveness of CPX in yeast provides a genetic system for identifying the target initiator protein.
- This approach facilitates the isolation of the mammalian homologue, aiding in the development of novel chemotherapeutics.