MOLECULAR MECHANISMS OF DDR GENE REGULATION IN YEAST

原核生物应激反应的分子特征 真核生物为基因机制提供了重要的新见解。 监管。两种典型的应激反应，热休克 反应和细菌‘SOS反应’提供了模型系统 了解多种基因元素如何转录 响应环境信号而被激活。中国人的DDR基因 单细胞真核生物酿酒酵母是一组基因，它们是 转录上对DNA损伤有反应。此外，这些基因中的三个 (DDRA2、DDR48和UBI4)在酵母中也能被热休克诱导。这个 DDR基因转录调控的分子特征将是 研究目的是：i)鉴定顺式作用序列 DNA损伤应激和热休克诱导的充要条件 转录；ii)纯化负责转录的反式作用因子 在胁迫后调节转录；以及iii)分离突变 参与应激反应途径的基因并克隆这些基因 元素。启动子融合将使用以下上游序列构建 基于缺失分析的DDR基因对应激很重要 调节，和一个异源的CyC-LacZ报告基因，以便准确地 定义那些同时导致DNA损伤和热休克的DNA序列 响应性。这些启动子融合构建体将用于分离 改变CyC-LacZ报告基因调控的突变 DNA损伤或热应激。凝胶延迟实验将用于 识别与这些重要调控区域结合的蛋白质。这些 实验将提供以下问题的答案：i)做DNA 损伤和热休克应激通过共同调控DDR基因表达 上游控制元素？二)是相同的反式作用因子 对DNA损伤和热休克反应负责吗？三)解甲返乡 基因协调调控？以及iv)有多少基因参与了 DDR基因的应激反应调控？这些研究将提供 对基因调控分子机制的重要见解 并有助于我们对分子反应的了解 细胞对诱变剂/致癌物的暴露。

The molecular characterization of stress responses in prokaryotes and eukaryotes has provided important new insights into the mechanisms of gene regulation. Two well characterized stress responses, the heat shock response and the bacterial 'SOS response' have provided model systems for understanding how multiple genetic elements can be transcriptionally activated in response to environmental signals. The DDR genes of the unicellular eukaryote Saccharomyces cerevisiae are a set of genes which are transcriptionally responsive to DNA damage. Moreover, three of these genes (DDRA2, DDR48 and UBI4) are also induced by heat shock in yeast. The molecular features of transcriptional regulation of the DDR genes will be investigated in order to: i) identify the cis-acting sequences that are necessary and sufficient for DNA damage stress and heat shock induction of transcription; ii) purify the trans-acting factors that are responsible for modulating transcription following stress; and iii) isolate mutations in genes that participate in the stress response pathways and to clone these elements. Promoter fusions will be constructed using upstream sequences of the DDR genes which, based upon deletion analysis are important for stress regulation, and a heterologous CYC-LACZ reporter gene in order to precisely define those DNA sequences that confer both DNA damage and heat shock responsiveness. These promoter fusion constructs will be used to isolate mutations that alter the regulation of the CYC-LACZ reporter gene following DNA damage or heat stress. Gel retardation experiments will be employed to identify proteins which bind to these important regulatory regions. These experiments will provide answers to the following questions: i) Do DNA damage and heat shock stress regulate DDR gene expression through common upstream control elements? ii) Are identical trans-acting factors responsible for both DNA damage and heat shock responses? iii) Are the DDR genes coordinately regulated? and iv) How many genes participate in the stress response regulation to the DDR genes? These studies will provide important insights into the molecular mechanisms of gene regulation in eukaryotes and contribute to our knowledge of the molecular responses of cells to mutagen/carcinogen exposure.