Eukaryotic cells have elaborate genome surveillance and repair machinery that insures accurate and timely execution of cell-cycle events, such as DNA replication. Defects in this machinery lead to genetic instability and increased cancer susceptibility. Replication stress is induced by damaged DNA, which stalls movement of DNA replication forks. This activates the 'replication checkpoint'to transmit signals that delay S-phase progression, and stabilize stalled forks so that DNA replication can resume after repair of the initiating lesion. The protein kinases, ATR and its downstream target, Chk1 are key components in this pathway. Loss of ATR or Chk1 function is lethal in both normal and malignant cells, underscoring the importance of the replication checkpoint in maintaining genome integrity. Several clinically useful anticancer agents (e.g. camptothecins (CRTs), target DNA replication, and are activators of the replication checkpoint. Preliminary studies of CPT-induced cytotoxicity in cancer cells led to the unexpected discovery that drug-treated cells had reduced Chk1 protein. Other agents (e.g., deep hypoxia, MMS or ionizing radiation) that cause fork stalling also elicit Chk1 downregulation. Chk1 degradation depends on cullin-containing E3 ubiquitin ligases;Chk1 destruction leads to irreversible replication arrest, DNA damage, and cell death. Defects in Chk1 degradation confer resistance to the cytotoxic effect of CPT - a major problem with this class of drugs in the clinic. This novel Chk1 regulation mechanism has important implications for our understanding of S-phase checkpoint signaling, as well as mechanisms of anticancer resistance in cancer patients. We propose to elucidate the underlying mechanisms and significance of stress-induced Chk1 destruction as follows: (1) Identify components of the E3 ligase complexes responsible for Chk1 destruction in cells exposed to replication stress;(2) Identify the putative 'degron'in Chk1;(3) Characterize mechanisms underlying defects in Chk1 degradation in CPT-resistant cancer cells;(4) Determine if failure to degrade Chk1 increases cancer cell resistance to hypoxia and alters response to chemo/radio-therapeutic agents. Relevance: The results of these studies will not only advance our understanding of the genotoxic stress response machinery in human cells, but also provide important insights into the antitumor mechanism of an important class of anticancer agents.
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