INTERACTION OF YEAST CHECKPOINT PROTEINS AS DRUG ASSAY

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Transient arrest of the eukaryotic cell cycle is an actively regulated response to DNA-damaging agents and other agents perturbing normal cell cycle progression, such as replication inhibitors or poisons of the mitotic spindle. Such checkpoints are quite frequently compromised in tumor cells and checkpoint failure may constitute an important factor contributing to the genetic instability that is commonly associated with the establishment of malignancy. Differential checkpoint responses in tumor cells vs. wild type cells may also translate into differential drug sensitivities since the majority of effective anticancer drugs are DNA-damaging agents or other agents that will induce a checkpoint arrest response. Rad17 of budding yeast Saccharomyces cerevisiae is an evolutionary conserved protein with an indispensable role in checkpoint arrest in G1 and G2 in response to DNA damage. Analysis of Rad17 in the yeast-two hybrid system suggested an increased homomeric complex formation in the presence of DNA damage. Such an effect can be demonstrated in a technically simple gradient assay performed on plates (DIPI assay, for DNA- damage-induced protein interaction). It is proposed to explore this system further with the dual goal of providing a screening system for high-throughput profiling of the checkpoint-activating or -modifying potential of candidate anticancer agents and at the same time elucidating the molecular basis of the Rad17 protein-protein interactions. We intend... To confirm a stimulation of Rad17/Rad17 complex formation by DNA damage through biochemical analysis. To identify regions of the Rad17 protein that are required for self- interaction and its regulation by DNA damage and to analyze the phenotype conferred by alterations in such regions. To develop the gradient assay ("DIPI assay") into a useful profiling tool by investigating the outcome for various substances, by comparing the assay to an established test for genotoxicity and by determining the influence of different mutant backgrounds.