ATR, a human phosphatidylinositol 3-kinase-related kinase, is an important component of the cellular response to DNA damage. In the present study, we evaluated the role of ATR in modulating the response of cells to S phase-associated DNA double-stranded breaks induced by topoisomerase poisons. Prolonged exposure to low doses of the topoisomerase I poison topotecan (TPT) resulted in S phase slowing because of diminished DNA synthesis at late-firing replicons. In contrast, brief TPT exposure, as

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... ell as prolonged exposure to the topoisomerase II poison etoposide, resulted in subsequent G 2 arrest. These responses were associated with phosphorylation of the checkpoint kinase Chk1. The cell cycle responses and phosphorylation of Chk1 were markedly diminished by forced overexpression of a dominant negative, kinase-inactive allele of ATR. In contrast, deficiency of the related kinase ATM had no effect on these events. The loss of ATR-dependent checkpoint function sensitized GM847 human fibroblasts to the cytotoxic effects of the topoisomerase I poisons TPT and 7-ethyl-10hydroxycamptothecin, as assessed by inhibition of colony formation, increased trypan blue uptake, and development of apoptotic morphological changes. Expression of kdATR also sensitized GM847 cells to the cytotoxic effects of prolonged low dose etoposide and doxorubicin, albeit to a smaller extent. Collectively, these results not only suggest that ATR is important in responding to the replication-associated DNA damage from topoisomerase poisons, but also support the view that ATM and ATR have unique roles in activating the downstream kinases that participate in cell cycle checkpoints. ATR 1 has been identified as one of the protein kinases that transduces signals to the cell cycle machinery during normal DNA replication (1) and after DNA damage (2-4). Like the structurally related kinases human ATM, Schizosaccharomy-ces pombe Rad3 (Rad3 SP ), and Saccharomyces cerevisiae Mec1 (Mec1 Sc ), ATR contains a conserved C-terminal kinase domain that phosphorylates downstream substrates (5). The nature of the DNA damage that activates ATR, the identity of its substrates, and the impact of ATR on cell cycle progression are currently the subject of extensive investigation. Previous studies have suggested that ATR and ATM might have distinct but overlapping functions. In response to IR, ATR has been observed to phosphorylate and activate the checkpoint kinase Chk1 (4) , which in turn phosphorylates Cdc25c, inactivating its phosphatase activity and contributing to the ensuing G 2 arrest (6 -8). In contrast, ATM, which appears to play the more critical role in response to IR, phosphorylates Chk2 (1, 9, 10). Despite these differences, Chen et al. (11) observed that Chk1 overexpression can complement the G 2 /M checkpoint defect in AT cells and restore IR resistance. These results suggest redundancy and overlap in the specific roles of ATM and ATR. Several observations indicate that ATM and ATR are also important in the intra-S checkpoint, a series of biochemical reactions that inhibit DNA synthesis in the face of DNA damage or stalled replication forks (12-15). ATM has been shown recently to initiate signaling through Chk2 and Cdc25A to inhibit Cdk2 and prevent DNA synthesis after IR (1). The response of AT cells to other inhibitors of DNA replication, however, appears intact despite the absence of functional ATM (10). Moreover, the observation that several proteins known to be regulated by ATM can still be activated by IR in AT cells (10) indicates that at least one additional upstream regulatory protein can initiate the S phase checkpoint. The possibility that ATR might play this role was initially suggested by the observation that ATR inhibition results in hypersensitivity to the replication inhibitors hydroxyurea and aphidicolin (2). Subsequent results indicated that ATR phosphorylates Chk1 in response to hydroxyurea (4). More recent experiments demonstrated that Xenopus ATR associates with chromatin in a replication-dependent manner, whereas ATM and DNA-PK do not (15). Interestingly, depletion of ATR in a cell-free Xenopus replication system blocked the Chk1 phosphorylation that ordinarily occurs after treatment with inhibitors of DNA replication (15). Collectively, these observations suggest that ATR is poised to respond to DNA damage occurring specifically during S phase. In contrast to IR, which induces multiple types of DNA damage throughout the cell cycle (16), the topo I poison CPT and its derivatives produce a single type of DNA lesion that is largely replication-dependent (17-21). Early studies demonstrated that CPT is selectively toxic during S phase (22) (23) (24) (25) . Subsequent investigations demonstrated that this S phase selectivity reflects the formation of DNA ds breaks when advancing replication forks collide with drug-stabilized topo I-DNA complexes (26 -29). Additional studies revealed that brief ex-