REPAIR OF DNA DAMAGED BY UV IRRADIATION IN YEAST

我们的具体目标是描述分子机制和遗传 控制紫外线（UV）损伤的DNA的细胞修复 在真核生物中，酵母Saccharomyces gouleae。 之间 参与UV诱导的p]嘧啶二聚体去除的突变体，切割 突变体与切除突变体的区别在于， 紫外线照射后DNA单链断裂的积累 切除和连接酶缺陷的RAD CDC 9双突变体。 休息时间将是 通过从原生质球裂解获得的DNA沉降测量， 直接在碱性蔗糖梯度上分层。 的程度 UV刺激的修复复制将通过密度标记来测量， 脱氧溴尿苷一磷酸（dBrUMP）。 我们将 纯化并研究了5'大于3'核酸外切酶和DNA连接酶， 涉及切除修复的酶活性，来自野生型和 各种修复突变体 复制后修复的研究将包括 确定重组的作用，哪些基因是后所需的 复制修复，以及它是否发生在线粒体DNA中。 RAD6 基因，在紫外线损伤的复制后修复中起重要作用 在紫外诱变中，将通过互补紫外辐射的rad 6 -1进行克隆 阻力 然后，我们将鉴定、纯化和表征RAD 6 蛋白质，以便了解它在DNA中的功能 修复. 为了确定是否影响紫外诱变的相同基因， 核DNA也影响线粒体DNA的紫外诱变，我们将 测定线粒体红霉素抗性的UV诱导频率 野生型突变体和UV诱变缺陷的各种修复突变体 核基因。 几种人类遗传疾病与 缺陷的DNA修复和增强的肿瘤转化。 的透彻 对DNA修复的分子机制的理解可能会提供一个 更好地了解致癌的原因。

Our specific aims are to characterize the molecular mechanisms and genetic control of cellular repair of DNA damaged by ultraviolet light (UV) irradiation in the eukaryote, the yeast Saccharomyces cerevisieae. Among mutants involved in removal of UV induced p]yrimidine dimers, incision mutants will be distinguished from excision mutants by monitoring the accumulation of single strand breaks in DNA following UV irradiation in excision and ligase defective rad cdc9 double mutants. Breaks will be measured by sedimentation of DNA obtained from spheroplasts lysed by layering directly on alkaline sucrose gradients. The extent of UV-stimulated repair replication will be measured by density labeling with deoxybromouridine monophosphate (dBrUMP) in tup tmp strains. We will purify and study 5' greater than 3' exonuclease and DNA ligase, two enzymatic activities involved in excision repair, from both wild type and various repair mutants. Studies of post-replication repair will include determining the role of recombination, which genes are required for post replications repair, and whether it occurs in mitochondiral DNA. The RAD6 gene, which plays an important role in post-replication repair of UV damage and in UV mutagenesis, will be cloned by complementation of rad6-1 for UV resistance. We will then identify, purify and characterize the RAD6 protein in order to gain some insight as to how it functions in DNA repair. For determining if the same genes that affect UV mutagenesis of nuclear DNA also affect UV mutagenesis of mitochondrial DNA, we will measure the UV induced frequency of mitochondiral erythromycin resistant mutants in wild type and various repair mutants defective in UV mutagenesis of nuclear genes. Several human genetic diseases are associated with defective DNA repair and enhanced neoplastic transformation. A thorough understanding of the molecular mechanisms of DNA repair may provide a better understanding of the causes of carcinogenesis.