INTERACTION OF YEAST CHECKPOINT PROTEINS AS DRUG ASSAY

真核细胞周期的瞬时停滞是对DNA损伤剂和干扰正常细胞周期进程的其他试剂（如复制抑制剂或有丝分裂纺锤体的毒物）的主动调节反应。 这样的检查点在肿瘤细胞中相当频繁地受损，并且检查点失败可能构成促成通常与恶性肿瘤的建立相关的遗传不稳定性的重要因素。 肿瘤细胞与野生型细胞中的差异检查点应答也可以转化为差异药物敏感性，因为大多数有效抗癌药物是DNA损伤剂或将诱导检查点停滞应答的其他试剂。Rad 17是芽殖酵母Saccharomycescerevisiae的一个进化保守蛋白，在DNA损伤后G1和G2期的检查点阻滞中起着不可或缺的作用。Rad 17在酵母双杂交系统中的分析表明，在DNA损伤的存在下，增加了同聚体复合物的形成。 这种效应可以在平板上进行的技术上简单的梯度测定（DIPI测定，用于DNA损伤诱导的蛋白质相互作用）中证明。建议进一步探索该系统，其双重目标是提供用于检查点激活或修饰候选抗癌剂的潜力的高通量分析的筛选系统，并同时阐明Rad 17蛋白质-蛋白质相互作用的分子基础。我们打算通过生化分析确认DNA损伤对Rad 17/Rad 17复合物形成的刺激。 鉴定Rad 17蛋白自身相互作用及其通过DNA损伤调节所需的区域，并分析这些区域改变所赋予的表型。 通过研究各种物质的结果，将梯度试验（“DIPI试验”）与已建立的遗传毒性试验进行比较，并确定不同突变体背景的影响，将其开发为有用的特征分析工具。

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.