In response to DNA damage, eukaryotic cells stop dividing and arrest in the G1 and G2 phases of the cell cycle. These arrests allow repair of the damaged genetic material prior to its replication in S phase and its distribution to the progeny in mitosis. An understanding of the mechanisms employed by cells to achieve these arrests may prove useful in defining new molecular targets for the treatment of malignancies. The transcriptional regulation of gene expression by p53 appears to be the primary mechanism used by cells to undergo a G1 arrest following DNA damage. p53-independent changes in gene expression are likely to participate in the regulation of the G2 DNA damage checkpoint, as evidenced by the down-regulation of cyclin B1 expression in p53- deficient cells. In an effort to better characterize the p53- independent changes in gene expression that follow DNA damage, DNA chip analysis has been performed using p53-deficient cells. Preliminary data has revealed four genes (cyclin B1, plk1, TTK, and p55cdc) that are down-regulated by gamma-irradiation, each of which has a proposed role in regulating entry into or progression through mitosis. Experiments are planned to better characterize the role of these proteins in entry into mitosis, and the relevance of their down-regulation in the G2 DNA damage checkpoint. In addition, experiments have been planned to identify other genes that are regulated by DNA damage, and to begin a characterization of the signaling pathway that leads to changes in gene transcription. The following specific aims are proposed. 1. Identify additional human genes that are induced or repressed by DNA damage during the G2 phase of the cell cycle in p53-deficient cells. 2. Analyze the role of proteins encoded by up- and down-regulated genes for a possible regulatory role in the G2 DNA damage checkpoint. 3. Characterize the mechanism leading to induction or repression of gene expression for selected genes following DNA damage. Identify the promoter element(s) responsible for the DNA damage responsive expression for one or more transcriptionally regulated genes.
Specific Aim number 3 is a conditional aim, and will not be pursued until the first two aims are completed. This will provide an understanding of the G2 DNA damage checkpoint.
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