DNA POLYMERASE ACCESSORY PROTEINS FROM EUKARYOTIC CELLS

准确的DNA复制对长期生存至关重要。 而 合成DNA所需的核心催化机制 在原核生物中以及在较小程度上在真核生物中具有特征， 提高复制效率的组件收到的 关注 虽然这些因素是必要的忠实基因组 重复，很难使用传统方法进行研究，因为 它们对无细胞检测和遗传学只有微妙的影响， 扰动很难从机械上解释。 这项建议 探讨了利用关联的方法的有用性， 蛋白质来识别和表征复制的组分 复合物在酵母酿酒酵母。 酵母DNA聚合酶α已经过表达、纯化并用作 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被保留在提取物中， 含有共价连接Polalpha分子的基质。 结合 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。 一个人的属性 这种结合蛋白已经通过体外测定和 研究突变的表型后果。 至少四 已经鉴定出另外的聚合酶结合蛋白， 表征了 还将使用利用蛋白质-蛋白质相互作用的其他方法。 已经制备了针对复制蛋白的抗体， 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 聚合酶基因将使用现有的修饰进行研究。 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的鉴定和表征将使用 “双杂交”方法，其中蛋白质-蛋白质相互作用连接DNA 识别元件转化为转录激活因子。 仅依赖于蛋白质之间的相互作用来重建 复合物可以揭示有关必需辅助因子的信息， 重要的保真度因素。 这些方法将用于 表征真核细胞中DNA复制的方面， 对传统方法来说很难。

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.