(From Abstract) Cell cycle checkpoints are surveillance mechanisms that induce growth arrest responses to ensure genetic fidelity and stability. Absence of checkpoint function can be associated with increased sensitivity to ionizing radiation (IR) and with genetic diseases such as the cancer prone disease ataxia telangiectasia that is caused by mutations within the ATM gene. The genes required for yeast checkpoint functions are well known but have not been fully characterized at the biochemical level. Human homologs for these genes have now been identified and include the main effector proteins kinases ATM and ATR; a complex of proteins believed to act upstream of ATM and ATR (HuRad1, HuHus1, HuRad9, and HuRad1; and downstream effector kinases HuChk1 and HuCds1 (Chk2) that appear to mediate growth arrest in G2 by targeting cdc25C. It has been shown that HuCds1 is activated in an ATM-dependent manner in response to IR. Experiments will be performed to determine whether ATM targets HuCds1 in response to IR and whether HuCds1 is a major effector of ATM-controlled pathways. Other mechanisms for the regulation of HuCds1 in response to DNA damage will be explored by determining whether other proteins bind to the fork head associated (FHA) domain of HuCds1 and modulate its activity in response to DNA damage. To begin to understand the functions of HuRad1, HuHus1, HuRad9, and HuRad17, dominant negative inhibitors will be designed to disrupt HuRad17 function and the HuRad1, HuHus1, and HuRad9 complex. These inhibitors will be tested as to whether their expression in cells interferes with the regulation of ATM kinase in response to IR and produce AT-like checkpoint phenotypes. Defining the molecular functions of these genes will not only improve our understanding of cell cycle regulation following DNA damage, but may also identify new gene products that could be targeted for the development of radiosensitizers.
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