CELL CYCLE RESPONSE TO RADIATION DAMAGE IN YEAST

总体目标是阐明细胞周期的机制。 处理后酿酒酵母中的细胞停滞 具有紫外线和电离辐射，特别强调大自然 检查点激活DNA损伤及其机制的研究进展 承认。真核细胞有能力可逆地 延缓细胞周期进程对辐射损伤的反应 离散过渡点称为检查点。据推测 检查站逮捕可以为DNA修复提供时间，以避免 以突变或突变的形式不可逆转地固定损伤 染色体异常。因此，对检查点的说明 它们在癌细胞中失活的机制及分析 对于理解细胞的过程很重要 遗传不稳定癌的转化与表型 细胞。遗传高度顺应性酵母 酿酒酵母已经成功地成为了 建立细胞周期检查点的概念。各种突变体 已知在61个、62个和5个阶段检查点有缺陷。它 提出用来分析辐射诱导细胞的机制 通过不同的互补方法阻止酵母中的循环。这个 具体目标如下：1.活动的特点 参与识别所需的DNA相互作用 检查点激活伤害。这包括提纯和 RAD17，一种可能参与3‘&gt；5’的核酸外切酶的特性 检查点截获和人类肿瘤的潜在酵母同源物 抑制子和检查点决定因素P53。此外，一个 蛋白质与候选DNA底物结合的分析将 提供有关细胞周期停滞的性质的信息- 触发DNA结构。2.筛选相互作用的蛋白质 与已知的检查点控制基因产物Rad17-和Mec1。 该方法包括成熟的酵母双杂交 多拷贝抑制基因和表达选择系统 His标记的检查站控制蛋白。基因 相互作用的蛋白质将被灭活，并且 将对产生的突变进行分析。3.选择和 G1期停滞缺陷酵母新突变体的特性研究。4.A 通过筛选检查点控制酵母基因的人类同源物 基于聚合酶链式反应的酵母功能互补策略 与人cDNA库的突变体。此外，潜在的 酵母RAD17和人P53的功能同源性将是 在互补性研究中探索。

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.