ENZYMATIC MECHANISMS OF DNA REPLICATION--THE BACTERIOPHAGE T4 SYSTEM

DNA复制的酶促机制--噬菌体T4系统

• 批准号: 3839741
• 负责人: N G NOSSAL
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3839741
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA primase; DNA replication; Escherichia coli; X ray crystallography; bacteriophage T4; crosslink; enzyme complex; enzyme mechanism; enzyme structure; gel mobility shift assay; gene deletion mutation; gene mutation; helicase; intermolecular interaction; mutant; nucleic acid sequence; protein purification; ribonuclease III; site directed mutagenesis; virus genetics; virus protein

We are continuing our study of the E. coli bacteriophage T4 model system for duplex DNA replication in which efficient DNA replication in vitro is achieved with purified proteins encoded by T4 phage: T4 DNA polymerase (gene 43), gene 32 DNA helix-destabilizing protein, the gene 44/62 and gene 45 polymerase accessory proteins, the genes 41, 61, and 59 primase- helicase, RNase H, and DNA ligase. Assembly of the polymerase and accessory proteins on the primer template. We have previously used cross-linking of proteins to photo-activatable residues at specific nucleotides in the primer to determine the position of polymerase and each of the accessory proteins on the primer. In order to position these proteins on the template strand, and to study the steps in the assembly of the complex by a gel-mobility shift assay, we are conducting a systematic analysis to determine the minimum size primer- template that will allow the proper assembly of the complex. Construction and analysis of mutations in T4 DNA polymerase. T4 DNA polymerase has extensive amino acid sequence similarity with a large family of prokaryotic and eukaryotic DNA polymerases. We are using in vitro mutagenesis to determine the function of these conserved regions and to try to identify regions of the polymerase important for its interaction with the other T4 replication proteins. We have constructed a polymerase lacking the editing exonuclease by changing a conserved amino acid in the N-terminal region. This mutant retains its polymerase activity and interacts normally with the accessory proteins, T4 phage with the exonuclease defective polymerase gene have a very high rate of mutation in vivo. Function of the T4 gene 59 helicase assembly factor. The addition of 59 protein to the other seven replication proteins gives a marked increase in synthesis on duplex DNA. We are currently studying how and where this synthesis is initiated. T4 RNaseH. We have identified a T4 gene encoding an RNaseH, and shown that the purified protein removes RNA primers in vitro. We have constructed a phage mutant with a deletion within the RNaseH gene, and shown that this gene is essential in a host that is lacking both RNaseH and 5 to 3 exonuclease of Pol I. Structure of the T4 replication proteins. We have begun a collaboration with Craig Hyde, NIAMS, to try to crystallize these proteins.

我们正在继续研究E.大肠杆菌噬菌体T4模型系统 对于双链体DNA复制，其中体外有效的DNA复制是 用T4噬菌体编码的纯化蛋白质实现：T4 DNA聚合酶 (gene 43），基因32 DNA螺旋不稳定蛋白，基因44/62和基因 45聚合酶辅助蛋白，基因41，61和59引发酶- 解旋酶、RNA酶H和DNA连接酶。 聚合酶和辅助蛋白在引物模板上的组装。 我们先前已经使用蛋白质的交联来光活化 引物中特定核苷酸处的残基，以确定 聚合酶和引物上的每种辅助蛋白。 为了 将这些蛋白质定位在模板链上，并研究 通过凝胶迁移率变动分析组装复合物，我们 进行系统分析以确定最小尺寸的引物- 模板，这将允许正确的组装复杂。 T4 DNA聚合酶突变的构建和分析。 T4 DNA 聚合酶具有广泛的氨基酸序列相似性与一个大的家族 原核和真核DNA聚合酶。 我们在体外 突变以确定这些保守区域的功能，并尝试 为了鉴定聚合酶的区域， 其他的T4复制蛋白 我们构建了一种聚合酶 通过改变外切核酸酶中的保守氨基酸来缺乏编辑外切核酸酶。 N-末端区域。 该突变体保留了其聚合酶活性， T4噬菌体通常与辅助蛋白相互作用， 外切核酸酶缺陷型聚合酶基因突变率很高， vivo. T4基因59解旋酶组装因子的功能。 新增59名 与其他七种复制蛋白相比， 双链DNA合成。 我们目前正在研究如何以及在哪里， 开始合成。 T4 RNA酶H。 我们已经鉴定了编码RNaseH的T4基因，并表明， 纯化的蛋白质在体外去除RNA引物。 我们已经建立了一个 在RNaseH基因内缺失的噬菌体突变体，并表明这 基因在缺乏RNaseH和5 - 3的宿主中是必需的 Pol I核酸外切酶。 T4复制蛋白的结构。 我们开始了一项合作 和NIAMS的克雷格·海德合作，试图使这些蛋白质结晶。