When cells suffer DNA damage cellular feedback or """"""""checkpoint"""""""" controls delay entry to mitosis and the initiation of DNA synthesis. We intend to determine the mechanism by which gamma-radiation-induced DNA damage results in such cell cycle delays in the fission yeast (Schizosaccharomyces pombe). The investigation will be in three parts. l. Our observations indicate that radiation-induced mitotic delay is imposed by inactivation of the mitotic kinase p34cdc2 We suggest that in addition to changes in its state of phosphorylation, down-regulation of p34 requires inactivation of cyclin (cdcl3+ product). This will be investigated by monitoring and manipulating levels of cdc13 product and message after irradiation. 2. Preliminary findings reveal an interaction between gene products which are required for the imposition of mitotic delay by irradiation (radl7 product) and the tumor suppressor protein p53 (when the latter is expressed in the fission yeast). The literature indicates that p53 may be required for the cell to delay DNA synthesis in response to irradiation. We will therefore a) determine whether radl7-W (and similar checkpoint mutants) fail to delay DNA synthesis initiation after irradiation and, b) characterize other gene products, identified by genetic procedures, which interact with the tumor suppressor protein. This approach may allow elucidation of both the mechanism of DNA synthesis delay and the function of p53. 3. To date no attempt has been made to identify conditional checkpoint mutants. Therefore, mutations in checkpoint control elements which are essential for viability are under-represented in known mutant series. Temperature-sensitive checkpoint mutants will be sought. Mutants will allow further genetic and molecular genetic characterization of the DNA damage checkpoint control. Use of the fission yeast, which has been extensively characterized for G2 cell cycle controls and checkpoint controls, allows a molecular genetic approach to this investigation. These studies will contribute to an understanding of cell cycle controls and their involvement in radiation responses and, conceivably, oncogenesis.
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