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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.
Abstract:
Inhibitors of DNA replication in mammalian cells are of great interest because of their potential use in chemotherapy and in cell synchronizing protocols in the laboratory. We have used a combination of isotopic labelling protocols and a two-dimensional gel replicon mapping procedure to determine the specific effects of five different replication inhibitors in cultured cells. Utilizing this protocol, we show that hydroxyurea, aphidicolin, and cytosine arabinoside, three known chain elongation inhibitors, are rather ineffective at preventing fork progression even at relatively high concentrations. In contrast, two related compounds that have been suggested to be G1/S inhibitors (mimosine and ciclopyrox olamine [CPX]) actually appear to inhibit initiation at origins. One of these agents (CPX) appears also to inhibit replication in yeast, opening the possibility that the gene encoding the target (initiator?) protein can first be identified in yeast by genetic approaches and can then be used to isolate the mammalian homologue.
Insights
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.