A general protocol for evaluating the specific effects of DNA replication inhibitors.
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.
University of Virginia School of Medicine, Department of Biochemistry, Charlottesville 22908.
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Levenson
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Hamlin
D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D016590:Aphidicolin / Q000494:pharmacology; D016466:CHO Cells; D002453:Cell Cycle / Q000187:drug effects; D000077768:Ciclopirox; D006224:Cricetinae; D003561:Cytarabine / Q000494:pharmacology; D004261:DNA Replication / Q000187:drug effects*; D015180:Electrophoresis, Gel, Two-Dimensional; D005434:Flow Cytometry; D005821:Genetic Techniques; D006918:Hydroxyurea / Q000494:pharmacology; D008898:Mimosine / Q000494:pharmacology; D011728:Pyridones / Q000494:pharmacology; D012093:Replicon / Q000187:drug effects; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics
D000935:Antifungal Agents; D011728:Pyridones; D003561:Cytarabine; D000077768:Ciclopirox; D016590:Aphidicolin; D008898:Mimosine; D013762:Tetrahydrofolate Dehydrogenase; D006918:Hydroxyurea