The Ataxia Telangiectasia protein kinase (ATM) is activated by the radiomimetic agent bleomycin and is required for cells to survive bleomycin-induced DNA damage. The signal transduction pathway which links the detection of bleomycin-induced DNA damage to ATM activation is poorly characterized. We have identified a novel 64 amino-acid N-terminal domain of the ATM protein which is an essential component of this signal transduction pathway. This 64 aminoacid domain of ATM is required for cells to survive bleomycin-induced DNA damage. The hypothesis to be tested is that this N-terminal domain of ATM is required for activation of ATM following exposure to bleomycin.
Aim 1. Deletion analysis and site-directed mutagenesis will be used to identify the exact amino-acids which constitute this domain. ATM clones will be constructed with this N-terminal domain inactivated, and cell lines stably expressing these clones will be prepared. The cells will then be exposed to bleomycin, and the role of the N-terminal regulatory domain of ATM in regulating cell survival and ATM kinase activity will be determined. Cell lines expressing the ATM construct in which the N-terminal is inactivated will be exposed to bleomycin. The ability of this ATM construct to activate cell cycle checkpoints and key ATM targets, including chk2 and p53, will be determined.
Aim 2. Proteins, which interact with the N-terminal of ATM will be identified by Mass Spectrometry. The functional role of these proteins in regulating ATM kinase activity and bleomycin sensitivity will be determined. Potential phosphorylation sites will be identified to determine how phosphorylation controls ATM activation. The identification of the exact position of this crucial N-terminal protein domain will elucidate the molecular basis by which bleomycin causes DNA damage in tumor cells and associated toxicity in normal tissue; it will contribute to the design of improved antibiotics; and it will identify new molecular targets for ATM inhibition.
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