MOLECULAR MECHANISM OF DDR GENE REGULATION IN YEAST

在细菌大肠杆菌中，一种复杂而协调的对 化学诱变剂治疗，被称为‘SOS’反应，涉及 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 单细胞真核生物酵母的应答(DDR) Cerevisiae，这可能是SOS调节子的对应物。我们 已经分离出一个由10多个基因组成的家族，这些基因显示出 在接触到各种不同的 诱变剂/致癌物。我们建议研究分子 其中两个基因DDRA2和DDRA2的调控细节 DDR48.为了鉴定DNA所需的序列 损害(和热休克)法规，将一连串的删除 使用去除增加的BAL31核酸外切酶构建 5‘上游区域的数量。这些因素的影响 关于文字记录规定的删除将由Northern进行评估 杂交分析。凝胶滞留分析将用于 研究蛋白质与重要上游区域的结合。 蛋白质-DNA接触的位置将由以下因素确定 在凝胶延迟的片段上执行‘足迹’。我们会 也检查围绕着染色质组织的变化。 利用DNAaseI研究DNA损伤或热休克后的DDR基因 过敏性实验。抗DDR基因抗体 将准备产品以调查本地化情况 受损细胞中的DDR蛋白。最后，我们会 DNA损伤对Ty转座刺激作用的研究 同时使用遗传和生化探针。

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.