The topoisomerase I poison camptothecin generates a Chk1-dependent DNA damage checkpoint signal in fission yeast.
The protein kinase Chk1 is essential for the DNA damage checkpoint. Cells lacking Chk1 are hypersensitive to DNA-damaging agents such as UV light and gamma-irradiation because they fail to arrest the cell cycle when DNA damage is generated. Phosphorylation of Chk1 occurs after DNA damage and is dependent on the integrity of the DNA damage checkpoint pathway. We have tested whether a topoisomerase I inhibitor, camptothecin (CPT), generates DNA damage in the fission yeast Schizosaccharomyces pombe that results in Chk1 phosphorylation. We demonstrate that Chk1 is phosphorylated in response to CPT treatment in a time- and dose-dependent manner and that phosphorylation is dependent on an intact DNA damage checkpoint pathway. Furthermore, we show that cells must be actively dividing in order for CPT to generate a Chk1-responsive DNA damage signal. This observation is consistent with a model whereby the cytotoxic event caused by CPT treatment is the production of a DNA double-strand break resulting from the collision of a DNA replication fork with a trapped CPT-topoisomerase I cleavable complex. Cells lacking Chk1 are hypersensitive to CPT treatment, suggesting that the DNA damage checkpoint pathway can be an important determinant for CPT sensitivity or resistance. Finally, as a well-characterized, soluble agent that specifically causes DNA damage, CPT will allow a biochemical analysis of the checkpoint pathway that responds to DNA damage.
Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854-5635, USA.
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Walworth
D002166:Camptothecin / Q000494:pharmacology*; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D004271:DNA, Fungal / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005800:Genes, Fungal; D009154:Mutation; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins; D015398:Signal Transduction; D059004:Topoisomerase I Inhibitors
D004271:DNA, Fungal; D004791:Enzyme Inhibitors; D029702:Schizosaccharomyces pombe Proteins; D059004:Topoisomerase I Inhibitors; D011494:Protein Kinases; D000071877:Checkpoint Kinase 1; C000605873:Chk1 protein, S pombe; D002166:Camptothecin