DNA POLYMERASE ACCESSORY PROTEINS FROM EUKARYOTIC CELLS

准确的DNA复制对长期生存至关重要。而当 合成DNA所需的核心催化装置已经很好地- 在原核生物中具有特征，在真核生物中的特征较少， 提高复制效率的组件收到的数量较少 请注意。而这些因素是忠诚的基因组所必需的 复制，它们很难用常规方法研究，因为 它们只会对无细胞检测和基因检测产生微妙的影响 微扰将很难从机械上解释。这项建议 探索利用两者之间的关联的方法的有用性 用于识别和表征复制组件的蛋白质 酿酒酵母中的复合体。 酵母DNA聚合酶α已经过表达，纯化，并用于 蛋白质亲和层析实验中的配体 与聚合酶相互作用的蛋白质通过一种 含有共价连接的Polpha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 携带这些基因突变的基因就会产生。One的性质 这种结合蛋白已经通过体外分析和 检查突变的表型后果。至少四个 更多的聚合酶结合蛋白已经被确定，并将被 特色化的。 还将使用其他利用蛋白质-蛋白质相互作用的方法。 针对复制蛋白的抗体已经制备出来，并将被 用于通过免疫共沉淀检测稳定的相互作用。遗传 两个基本DNA的温度敏感突变的抑制因子 将使用现有的一种修改来研究聚合酶基因 基于转座子的诱变过程。与DNA相互作用的蛋白质 体内的聚合酶α将通过使用 蛋白质-蛋白质相互作用连接DNA的“双杂交”方法 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 复合体可以揭示有关基本辅助因素和 关于重要的保真度因素。这些方法将用于 描述在真核细胞中DNA复制的特征 对传统方法来说是难以接受的。

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.