DNA POLYMERASE ACCESSORY PROTEINS FROM EUKARYOTIC CELLS

来自真核细胞的 DNA 聚合酶辅助蛋白

• 批准号: 2182000
• 负责人: Timothy G Formosa
• 金额: $ 17.53万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2182000
• 关键词: DNA binding protein; DNA damage; DNA directed DNA polymerase; DNA repair; DNA replication; Saccharomyces cerevisiae; affinity chromatography; eukaryote; fungal genetics; gene mutation; immunoprecipitation; intermolecular interaction; nucleic acid metabolism; nucleic acid sequence; protein structure function; temperature sensitive mutant

Accurate DNA replication is essential for long-term survival. While the core catalytic machinery needed to synthesize DNA has been well- characterized in prokaryotes and to a lesser extent in eukaryotes, components that promote efficiency of replication have received less attention. While such factors are necessary for faithful genome duplication, they are difficult to study using conventional methods since they would have only subtle effects on cell-free assays and genetic perturbations would be hard to interpret mechanistically. This proposal explores the usefulness of methods that exploit associations between proteins to identify and characterize the components of the replication complex in the yeast Saccharomyces cerevisiae. Yeast DNA polymerase alpha has been overexpressed, purified, and used as the ligand in protein affinity chromatography experiments in which proteins that interact with polymerase are retained from an extract by a matrix containing covalently attached Polalpha molecules. Binding proteins are sequenced, the genes encoding them are isolated, and cells carrying mutations in these genes are produced. The properties of one such binding protein has been studied both by in vitro assays and by examining the phenotypic consequences of mutations. At least four additional polymerase binding proteins have been identified and will be characterized. Other methods that exploit protein-protein interactions will also be used. Antibodies against replication proteins have been prepared and will be used to detect stable interactions by co-immunoprecipitation. Genetic suppressors of temperature-sensitive mutations in two essential DNA polymerase genes will be studied using a modification of an existing transposon-based mutagenesis procedure. Proteins that interact with DNA polymerase alpha in vivo will be identified and characterized using the "two hybrid" method in which the protein-protein interaction links a DNA recognition element to a transcriptional activator. Methods that rely only on the interactions between proteins to reconstruct complexes can reveal information about essential accessory factors and about important fidelity factors. These methods will be used to characterize aspects of DNA replication in a eukaryotic cell that have been refractory to traditional methods.

准确的 DNA 复制对于长期生存至关重要。 虽然 合成 DNA 所需的核心催化机制已被充分研究 以原核生物为特征，并在较小程度上以真核生物为特征， 促进复制效率的成分受到的影响较少 注意力。 虽然这些因素对于忠实的基因组是必要的 重复，因此很难使用传统方法进行研究，因为 它们只会对无细胞测定和遗传产生微妙的影响 扰动很难从机械上解释。 这个提议 探索利用之间关联的方法的有用性 蛋白质来识别和表征复制的组成部分 酿酒酵母中的复合物。 酵母 DNA 聚合酶 α 已过表达、纯化并用作 蛋白质亲和层析实验中的配体，其中 与聚合酶相互作用的蛋白质被保留在提取物中 含有共价连接的 Polalpha 分子的基质。 装订 蛋白质被测序，编码它们的基因被分离，细胞 这些基因携带突变就产生了。 一的属性 这种结合蛋白已通过体外测定和通过 检查突变的表型后果。 至少四个 其他聚合酶结合蛋白已被鉴定并将被 特点。 还将使用利用蛋白质-蛋白质相互作用的其他方法。 针对复制蛋白的抗体已制备完毕并将 用于通过免疫共沉淀检测稳定的相互作用。 遗传 两种必需 DNA 中温度敏感突变的抑制因子 聚合酶基因将通过现有技术的修饰进行研究 基于转座子的诱变程序。 与 DNA 相互作用的蛋白质 体内聚合酶α将使用以下方法进行鉴定和表征 “双杂交”方法，其中蛋白质-蛋白质相互作用连接 DNA 转录激活子的识别元件。 仅依靠蛋白质之间的相互作用来重建的方法 复合物可以揭示有关重要辅助因素的信息和 关于重要的保真度因素。 这些方法将用于 描述真核细胞中 DNA 复制的各个方面 传统方法难以解决。