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 l acking Chk1 are hypersensitive to DNA-damaging agents such as UV light and gamma-irradiation because they fail to arrest the cell cycle when DNA damag e is generated. Phosphorylation of Chk1 occurs after DNA damage and is depe ndent on the integrity of the DNA damage checkpoint pathway. We have tested whether a topoisomerase I inhibitor, camptothecin (CPT), generates DNA dam age in the fission yeast Schizosaccharomyces pombe that results in Chk1 pho sphorylation. 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-r esponsive DNA damage signal. This observation is consistent with a model wh ereby the cytotoxic event caused by CPT treatment is the production of a DN A double-strand break resulting from the collision of a DNA replication for k with a trapped CPT-topoisomerase I cleavable complex. Cells lacking Chk1 are hypersensitive to CPT treatment, suggesting that the DNA damage checkpo int pathway can be an important determinant for CPT sensitivity or resistan ce. Finally, as a well-characterized, soluble agent that specifically cause s DNA damage, CPT will allow a biochemical analysis of the checkpoint pathw ay that responds to DNA damage. Copyright (C) 1999 John Wiley & Sons, Ltd.