REPAIR OF DNA DAMAGED BY UV IRRADIATION IN YEAST

这项提议的长期目标是理解分子 紫外线损伤DNA模板在酵母中的旁路复制机制 酿酒酵母。色素性干皮病患者患有 皮肤癌的高发病率，可能是因为诱变旁路 DNA中未切除的紫外线损伤。拟议的项目将重点放在 复制后修复通路的主要无错误分支在 酵母。这条途径相当重要，因为它会产生一个 与诱变旁路相比，突变水平相对较低 路径。将进行遗传和生化研究，以确定 无错误旁路的各种组件，以定义它们的 生化活性，并在体外重建旁路复制。 复制后修复将在RAD5和增殖细胞核抗原的突变中进行检测 单独和与rev3Delta结合的基因。DNA的参与 β和epsilon聚合酶及单链结合蛋白RFA 复制后修复将通过遗传和分子来确定 学习。介导RAD6和RAD18蛋白相互作用的结构域 将被定义，并且相互作用肽的结构由 核磁共振波谱。Rad-18-ATPase在DNA结合和转运中的作用 将检测Rad6/Rad 18复合体的DNA结合活性 该综合体将进一步确定其特征。它在生物学上的意义 通过突变赖氨酸来确定三磷酸腺苷的结合和水解 Rad 18 GKS基序中的残基及其对DNA修复的影响 突变的rad18蛋白将被提纯并检测它们的能力 结合和水解三磷酸腺苷，以及DNA结合。Rad 18锌的作用 DNA结合中的指状域将通过定点突变进行检测 该基序的特定残基并测定其作用 这些rad18突变对DNA修复和DNA结合有影响。RAD5结合到 将检查不同的DNA底物，并将进行研究 确定是否将RAD6/RAD 18和RAD5加载到DNA复制上 受损DNA上的机器。RAD6、RAD18、RAD18的蛋白质相互作用 RAD5、增殖细胞核抗原和其他蛋白质在复制后间隙中起作用 填充将通过免疫共沉淀和亲和力进行研究。 层析，并由两种混合系统组成。编码其他基因 与Rad6、Rad18和Rad5相互作用的蛋白质将通过 二次混合法。这种方法应该会发现任何额外的蛋白质 可能需要旁路复制以及体内底物 RAD6。RAD6/RAD18复合体是否介导泛素化和 随后的降解或蛋白水解性切割特定的修复或 DNA损伤剂处理细胞后的复制蛋白 将会被确定。RAD+的无细胞提取物与多种修复 将检查缺陷突变体是否有紫外线辐射的旁路复制 DNA底物。绕过携带特定站点的模板的复制 紫外线损伤将使用纯化的成分进行重建。

The long term objectives of this proposal are to understand the molecular mechanisms of bypass replication of UV damaged DNA templates in the yeast Saccharomyces cerevisiae. Xeroderma pigmentosum patients suffer from a high incidence of skin cancers, presumably because of the mutagenic bypass of unexcised UV lesions from DNA. The proposed project will focus on the predominantly error-free branch of the postreplication repair pathway in yeast. This pathway is of considerable importance because it generates a relatively low level of mutations compared to the mutagenic bypass pathway. Genetic and biochemical studies will be done to identify the various components of the error free bypass pathway, to define their biochemical activities, and to reconstitute bypass replication in vitro. Postreplication repair will be examined in mutants of the RAD5 and PCNA genes, singly and in combination with rev3delta. The involvement of DNA polymerases delta and epsilon and of single strand binding protein RFA in postreplication repair will be ascertained by genetic and molecular studies. The domains that mediate interaction of Rad6 and Rad 18 proteins will be defined and the structure of interacting peptides determined by NMR spectroscopy. The role of Rad 18 ATPase in DNA binding and in turnover of the Rad6/Rad 18 complex will be examined, and DNA binding activity of the complex will be further characterized. The biological significance of ATP binding and hydrolysis will be ascertained by mutating the lysine residue in the GKS motif of Rad 18 and examining its effect on DNA repair. Mutant rad18 proteins will be purified and examined for their ability to bind and hydrolyze ATP, and for DNA binding. The role of the Rad 18 zinc finger domain in DNA binding will be examined by site directed mutagenesis of specific residues of this motif and determination of the effect of these rad18 mutations on DNA repair and on DNA binding. Binding of Rad5 to different DNA substrates will be examined, and studies will be done to determine if Rad6/Rad 18 and Rad5 are loaded onto the DNA replication machinery on damaged DNA. Protein-protein interactions among Rad6, Radl8, Rad5, PCNA, and other proteins found to have a role in postreplicative gap filling will be studied by coimmunoprecipitation, by affinity chromatography, and by the two hybrid system. Genes encoding other proteins that interact with Rad6, Rad18, and Rad5 will be cloned by the two hybrid method. This method should uncover any additional proteins that may be required for bypass replication, as well as the in vivo substrates of Rad6. Whether Rad6/Radl8 complex mediates the ubiquitination and subsequent degradation or proteolytic cleavage of specific repair or replication proteins following treatment of cells with DNA damaging agents will be determined. Cell-free extracts from RAD+ and various repair deficient mutants will be examined for bypass replication of UV irradiated DNA substrates. Bypass replication of templates carrying a site specific UV lesion will be reconstituted using purified components.