INTERACTION OF YEAST CHECKPOINT PROTEINS AS DRUG ASSAY

真核细胞周期的短暂停滞是对dna损伤剂和其他干扰正常细胞周期进程的药物（如复制抑制剂或有丝分裂纺锤体毒物）的主动调节反应。这些检查点在肿瘤细胞中经常被破坏，检查点失败可能是导致遗传不稳定的一个重要因素，而遗传不稳定通常与恶性肿瘤的形成有关。肿瘤细胞与野生型细胞的不同检查点反应也可能转化为不同的药物敏感性，因为大多数有效的抗癌药物是dna损伤剂或其他会诱导检查点阻滞反应的药物。酿酒酵母（Saccharomyces cerevisiae）的Rad17是一种进化保守蛋白，在DNA损伤的G1和G2阶段的检查点阻滞中起着不可或缺的作用。Rad17在酵母- 2杂交系统中的分析表明，在DNA损伤的情况下，同源复合体的形成增加。这种效应可以通过在平板上进行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）来证明。我们建议进一步探索这一系统，以提供一个高通量筛选系统来分析候选抗癌药物的检查点激活或修饰潜力，同时阐明Rad17蛋白-蛋白相互作用的分子基础。我们打算……通过生化分析证实DNA损伤对Rad17/Rad17复合物形成的刺激作用。鉴定Rad17蛋白的自相互作用和DNA损伤调控所必需的区域，并分析这些区域改变所赋予的表型。通过调查各种物质的结果，通过将分析与既定的遗传毒性测试进行比较，并通过确定不同突变背景的影响，将梯度分析（“DIPI分析”）发展成为一种有用的分析工具。

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.