MOLECULAR MECHANISMS OF DDR GENE REGULATION IN YEAST

原核生物胁迫反应的分子特征和 真核生物提供了重要的新的见解基因的机制， 调控两种典型的应激反应，热休克 反应和细菌的“SOS反应”提供了模型系统， 了解多个基因元件如何在转录水平上 响应环境信号。的DDR基因 单细胞真核生物酿酒酵母是一组基因， 对DNA损伤的转录反应。此外，其中三个基因 在酵母中，DDRA 2、DDR48和UBI 4也由热激诱导。的 DDR基因转录调控的分子特征将被 研究，以便：i）鉴定顺式作用序列， DNA损伤胁迫和热休克诱导的必要性和充分性 转录; ii）纯化负责转录的反式作用因子。 在应激后调节转录;和iii）分离 参与应激反应途径的基因， 元素启动子融合体将使用以下的上游序列构建： 基于缺失分析，DDR基因对于胁迫是重要的 调节，和异源CYC-LACZ报告基因，以便精确地 定义那些DNA序列，赋予DNA损伤和热休克 响应能力。这些启动子融合构建体将用于分离 突变改变CYC-LACZ报告基因的调节， DNA损伤或热应激。凝胶阻滞实验将用于 鉴定与这些重要调控区结合的蛋白质。这些 实验将提供以下问题的答案：i）DNA 损伤和热休克应激通过共同的途径调节DDR基因的表达 上游控制元件？2.相同的反式作用因子 导致DNA损伤和热休克反应㈢复员方案是否 基因协调调控？（4）有多少基因参与了 DDR基因对应激反应的调节作用这些研究将提供 对基因调控的分子机制的重要见解， 真核生物，并有助于我们的分子反应的知识， 细胞暴露于诱变剂/致癌物。

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.