MUTAGENESIS AND REPAIR OF DNA

该项目的长期目标是了解分子基础 紫外线和化学诱变，以了解E。大肠杆菌UmuD 和UmuC蛋白在这个过程中，并使用知识库 为该系统研究蛋白质结构功能问题积累了经验 蛋白质间的相互作用。 的分子机制 紫外线和化学诱变在任何生物体中都是未知的， 对理解化学致癌作用的基础和 一些人类遗传疾病的起源。 该提案利用了 的力量和相对容易的E。大肠杆菌遗传学，分子生物学， 生物化学来解决这个核心的未解决的问题， 生物学 生化方法将用于研究UmuC的影响， UmuD和其他蛋白质对DNA聚合酶在DNA上的行为 在已知位置携带确定的化学损伤的模板。 我们将 还试图确定UmuD和UmuC蛋白的功能， 寻找与T4噬菌体相似的活性 DNA聚合酶辅助蛋白和通过使用基于DNA的测定 携带大的反向重复序列的基质。 与Greg合作 Petsko和Dagmar林格的实验室，将努力 确定UmuD+的晶体结构。 UmuD的单半胱氨酸衍生物 和UmuD+将被构建并用于研究构象， 这些蛋白质的相互作用。 基因和生物化学技术将 用于分析UmuD和UmuC与其他蛋白质的相互作用。 一 将对一组突变UmuD和UmuD +蛋白进行生化分析， 确定它们关于同二聚体形成的缺陷的性质， 与RecA的相互作用、自身消化能力以及与UmuC的相互作用。 我们发现某些生理现象有可能 提供对UmuD和UmuC的见解，并将进一步分析;这些 包括抑制由增加剂量的 umuDC和umuDC依赖性DNA复制抑制。 我们将测试 在遗传学上，umuDC功能是否对细胞活力至关重要。

The long term goals of this project are to understand the molecular basis of UV and chemical mutagenesis, to understand the roles of the E. coli UmuD and UmuC proteins in this process, and to use the knowledge base accumulated for this system to study issues in protein-structure function relationships, and protein-protein interactions. The molecular mechanism of UV and chemical mutagenesis is not yet known in any organism yet is central to an understanding of the basis of chemical carcinogenesis and of the origin of some human genetic diseases. This proposal takes advantage of the power and relative ease of E. coli genetics, molecular biology, and biochemistry to address this central unsolved problem in nucleic acid biology. Biochemical methods will be used to study the influence of UmuC, UmuD, and other proteins on the behavior of DNA polymerases on DNA templates carrying a defined chemical lesion at a known position. We will also attempt to determine functions of the UmuD and UmuC proteins by looking for activities suggested by their similarity to bacteriophage T4 DNA polymerase accessory proteins and by using an assay based on DNA substrates carrying a large inverted repeat. In collaboration with Greg Petsko's and Dagmar Ringe's laboratories, an effort will be made to determine the crystal structure of UmuD+. Monocysteine derivatives of UmuD and UmuD+ will be constructed and used to study the conformation and interactions of these proteins. Genetic and biochemical techniques will be used to analyze the interactions of UmuD and UmuC with other proteins. A set of mutant UmuD and UmuD + proteins will be analyzed biochemically to determine the nature of their defects with respect to homodimer formation, interaction with RecA, autodigestion capability, and interaction with UmuC. Certain physiological phenomenon we have identified have the potential to offer insights into UmuD and UmuC and will be analyzed further; these include the inhibition of SOS-induction caused by an increased dosage of umuDC and umuDC-dependent inhibition of DNA replication. We will test genetically whether umuDC function is essential for cell viability.