MOLECULAR MECHANISM OF DDR GENE REGULATION IN YEAST

在E.大肠杆菌，一个复杂的和协调的反应， 化学诱变剂处理，称为“SOS”反应，包括 超过15个不同基因的转录改变。 我的实验室已经开始对DNA损伤进行分子分析 单细胞真核酵母中的DDR反应 这可能是一个对应的'SOS'调节子。 我们 已经分离出一个超过10个基因的家族， 转录后暴露于各种各样的 诱变剂/致癌物。 我们建议研究 这些基因中的两个，DDRA 2和 DDR48。 为了确定DNA所需的序列 损害（和热休克）调节，一系列删除将 使用BAL 31外切核酸酶构建，其去除增加的 5'上游区域的量。 成效为何 转录调控的缺失将由北方 杂交分析 凝胶阻滞试验将用于 研究蛋白质与重要上游区域的结合。 蛋白质-DNA接触的位点将通过 在凝胶阻滞的碎片上进行“足迹”。 我们将 还检查周围的染色质组织的变化， DNA损伤或热休克后的DDR基因 超敏反应实验 DDR基因抗体 产品将准备，以调查本地化 受损细胞中的DDR蛋白。 最后我们将 研究DNA损伤对Ty转座的刺激作用 使用基因和生化探针。

In the bacteriun E. coli, a complex and coordinate response to chemical mutagen treatment, termed the 'SOS' response, involves alterations of the transcription of more than 15 diverse genes. My laboratory has begun a molecular analysis of a DNA damage response (DDR) in the unicellular eucaryote Saccharomyces cerevisiae which may be a counterpart to the 'SOS' regulon. We have isolated a family of more than 10 genes which show altered transcription following exposure to a wide variety of mutagens/carcinogens. We propose investigating the molecular details of the regulation of two of these genes, DDRA2 and DDR48. In order to identify the sequences required for DNA damage (and heat shock) regulation, a series of deletions will be constructed using BAL31 exonuclease that remove increasing amounts of the 5' upstream regions. The effects of these deletions on transcript regulation will be assessed by Northern hybridization analysis. Gel retardation assays will be used to investigate binding of proteins to important upstream regions. The sites of protein-DNA contact will be determined by performing 'footprinting' on the gel retarded fragment. We will also examine changes in chromatin organization surrounding the DDR genes following DNA damage or heat shock using DNAaseI hypersensitivity experiments. Antibodies to the DDR gene products will be prepared in order to investigate the localization of the DDR proteins in damaged cells. Finally, we will investigate the stimulation of Ty transposition by DNA damage using both genetic and biochemical probes.