CELL CYCLE RESPONSE TO RADIATION DAMAGE IN YEAST

总体目标是阐明细胞周期的机制 在处理后的酵母酿酒酵母中的停滞 紫外线和电离辐射，特别强调自然 检查点激活的DNA损伤及其机制 识别.真核细胞具有可逆地 延迟细胞周期进程对辐射损伤的反应. 离散的转换点称为检查点。假设在 检查点阻滞可以为DNA修复提供时间， 以突变的形式不可逆转地固定损害，或 染色体畸变因此，检查点的说明 机制和分析其在癌细胞中的失活， 对于理解细胞的 转化和遗传不稳定癌症的表型 细胞遗传上高度顺从的酵母菌 酿酒酵母已经成功地作为模式生物， 建立细胞周期检查点的概念。各种突变体 已知在61、62和5阶段检查点中有缺陷。它 分析了辐射诱导细胞凋亡的机制 通过各种互补方法在酵母中进行周期阻滞。的 具体目标如下：1.活动的特点 参与DNA相互作用， 检查点激活损伤这包括净化和 Radl 7是一种假定的3 '> 5'外切核酸酶，参与了 检查点阻滞和人类肿瘤的潜在酵母同系物 抑制子和检查点决定子p53。此外， 分析蛋白质与候选靶DNA底物的结合将 提供关于细胞周期停滞性质的信息- 触发DNA结构2.蛋白质的筛选 与已知的检查点控制基因产物Rad 17-和Mec 1。 该方法包括成熟的酵母双杂交 系统，多拷贝抑制基因和表达的选择 控制检查点的蛋白质基因 相互作用的蛋白质将被灭活， 将分析所得突变体。3.选择和 G1停滞缺陷的新型酵母突变体的表征。4.A 筛选检查点控制酵母基因的人类同源物， 基于PCR的策略或通过酵母的功能互补 突变体与人cDNA文库。此外，潜在 酵母Rad 17和人p53之间的功能同源性将是 在互补研究中探索。

The overall goal is to elucidate the mechanisms of cell cycle arrest in the yeast Saccharomyces cerevisiae following treatment with UV and ionizing radiation, with special emphasis on the nature of the checkpoint-activating DNA damage and the mechanism of its recognition. Eukaryotic cells have the capability to reversibly delay cell cycle progression in response to radiation damage at discrete transition points termed checkpoints. It is assumed that checkpoint arrest can provide time for DNA repair in order to avoid irreversible fixation of damage in the form of mutations or chromosome aberrations. Thus, the elucidation of checkpoint mechanisms and an analysis of their inactivation in cancer cells is of importance for understanding the process of cellular transformation and the phenotype of genetically unstable cancer cells. The genetically highly amenable yeast Saccharomyces cerevisiae has already served successfully as a model organism to establish the concept of cell cycle checkpoints. Various mutants are known to be defective in 61, 62 and in 5-phase checkpoints. It is proposed to analyze the mechanisms of radiation-induced cell cycle arrest in yeast by various complementary approaches. The specific aims are as follows: 1.Characterization of activities involved in DNA interactions required for the recognition of checkpoint-activating damage. This includes the purification and characterization of Radl7, a putative 3'>5' exonuclease involved in checkpoint arrest and a potential yeast homolog of the human tumor suppressor and checkpoint determinant p53. Additionally, an analysis of protein binding to candidate target DNA substrates will provide information on the nature of the cell-cycle arrest- triggering DNA structures. 2.A screen for proteins that interact with the known checkpoint controlling gene products Rad17-and Mec1. The methodology includes the well-established yeast two-hybrid system, a selection for multi-copy suppressor genes and expression of His-tagged checkpoint-controlling proteins. Genes for interacting proteins will be inactivated and the phenotype of the resulting mutants will be analyzed. 3.Selection and characterization of novel yeast mutants defective in G1 arrest. 4.A screen for human homologs of checkpoint-controlling yeast genes by PCR-based strategies or by functional complementation of yeast mutants with human cDNA libraries. Additionally, potential functional homology between yeast Rad17 and human p53 will be explored in complementation studies.