REPAIR OF DNA DAMAGED BY UV IRRADIATION IN YEAST

长期目标是了解分子机制， 紫外线损伤DNA复制后修复的遗传调控 光（UV）在真核生物酿酒酵母中的作用。 以下 为达致这个目标，我们会进行研究。 RAD6基因， 所需的复制后修复，诱导诱变，和其他 重要的细胞功能将被随机诱变， 各种突变，并且还分离温度敏感（TS）突变体， 与rad6突变体相关的三种主要表型中的每一种：UV 敏感性、UV不变性和孢子形成缺陷。 位点特异 诱变将用于删除和修饰多酸区中的多酸区。 RAD6蛋白的碳基末端区域，以确定其功能。 RAD6和其他基因在复制后修复中的作用将被进一步研究。 从使用UV照射的单链DNA的体内实验评估 质粒以及体外实验来测量旁路 复制的 纯化的RAD 6蛋白将进一步表征为： 确定它是否与核小体相互作用。 由于RAD6基因是 在紫外线照射后以及在细胞周期中诱导，DNA 将确定其诱导所需的RAD 6基因序列。 粟酒裂殖酵母中的一种单一蛋白与抗体发生交叉反应 针对S.啤酒。 色葡萄粟酒基因 将克隆编码这种蛋白质的蛋白质，并将其编码的蛋白质 表征了 进一步扩展对药物代谢的分子机制的研究 复制后修复将包括分离和表征的 RAD18、REV3和RAD9基因，RAD6中的其他重要基因 上位组 由这些基因编码的蛋白质将被纯化， 研究了 为了实现界定《公约》各组成部分的最终目标， 参与复制后修复的蛋白复合物， 诱变、蛋白质或编码与RAD6相互作用的蛋白质的基因 蛋白质将直接使用遗传方法或 生物化学方法 遗传学方法将需要隔离寒冷 热敏UV敏感或UV不变性rad6的敏感抑制剂 变种人 生物化学方法将涉及免疫沉淀， 在复合物中与RAD6相关的蛋白质， 与RAD6结合的蛋白质。 此外，紫外线敏感性的抑制剂 或rad6缺失的UV不变性将被分离， 确定并表征抑制机制。 这种旁路 抑制可以发现可以克服rad6缺失的新基因 缺损 缺陷的DNA修复和增强的瘤形成表征了几种 人类遗传疾病。 彻底了解分子 DNA修复机制可能会提供更好的理解的原因， 致癌作用。

The long term objective is to understand the molecular mechanisms and genetic control of post-replication repair of DNA damaged by ultraviolet light (UV) in the eukaryote, Saccharomyces cerevisiae. The following studies will be carried out to achieve this goal. The RAD6 gene, which is required for postreplication repair, induced mutagenesis, and other important cellular functions will be randomly mutagenized to generate various mutations and also to isolate temperature sensitive (ts) mutants of each of the three major phenotypes associated with rad6 mutants: UV sensitivity, UV immutability, and sporulation deficiency. Site-specific mutagenesis will be used to delete and modify the polyacidic tract in the carbosyl-terminal region of the RAD6 protein to determine its function. The role of the RAD6 and other genes in postreplication repair will be assessed from in vivo experiments utilizing UV irradiated single-stranded plasmids as well as from in vitro experiments to measure bypass replication. The purified RAD6 protein will be further characterized by determining whether it interacts with nucleosomes. Since the RAD6 gene is induced following UV irradiation and also during the cell cycle, the DNA sequences in the RAD6 gene required for its induction will be determined. A single protein in Schizosaccharomyces pombe cross-reacts with antibody directed against the RAD6 protein of S. cerevisiae. The S. pombe gene encoding this protein will be cloned and the protein encoded by it will be characterized. Further extension of studies on the molecular mechanisms of postreplication repair will include isolation and characterization of the RAD18, REV3, and RAD9 genes, the other important genes in the RAD6 epistasis group. The proteins encoded by these genes will be purified and studied. For attaining the ultimate goal of defining the components of the protein complexes involved in postreplication repair and induced mutagenesis, proteins or genes encoding proteins which interact with RAD6 protein will be isolated directly using either a genetic approach or a biochemical approach. The genetic approach will entail isolation of cold sensitive suppressors of heat sensitive UV sensitive or UV immutable rad6 mutants. The biochemical approach will involve immunoprecipitation of proteins associated with RAD6 in a complex and affinity chromatography of proteins which bind to RAD6. In addition, suppresors of the UV sensitivity or UV immutability of rad6 deletions will be isolated and the genetic mechanism(s) of suppression identified and characterized. Such bypass suppression can uncover new genes which can overcome the rad6 deletion defect. Defective DNA repair and enhanced neoplasia characterize several human genetic diseases. A thorough understanding of the molecular mechanisms of DNA repair may provide a better understanding of the causes of carcinogenesis.