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: 3854688
• 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/3854688
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA primase; DNA replication; DNA replication origin; Escherichia coli; bacterial genetics; bacteriophage T4; crosslink; enzyme biosynthesis; enzyme mechanism; enzyme structure; gene mutation; intermolecular interaction; microorganism genetics; molecular cloning; mutant; protein purification; ribonuclease III; 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: these include 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. Using a system in which a photoactivatable aryl azide can be moved stepwise through a thirty base region of the primer, we find that the accessory proteins and 32 protein can be cross-linked to the primer in the presence of a non-hydrolyzable ATP analogue. The initial binding of the three accessory proteins and ATP to a 32 protein-covered primer-template is followed by ATP hydrolysis, binding of polymerase, and movement of each of the accessory proteins to yield a complex capable of processive DNA synthesis. Protein-protein interactions required for strand displacement synthesis. Mutations in the T4 DNA polymerase can decrease its ability to interact with the proteins that allow strand displacement synthesis with the wild type enzyme. The purified tsLl4l mutant polymerase (alanine changed to valine at 737) is stimulated by the accessory proteins and 32 protein, but is arrested at pause sites on single-stranded templates more frequently that the wild type, and carries out practically no strand displacement synthesis on a forked DNA template with the accessory proteins, 32 protein, and the 41 protein helicase. We have recently cloned and purified to homogeneity the T4 gene 59 protein, which increases DNA unwinding by the 41 helicase. We find that addition of this 59 protein is absolutely required for strand displacement synthesis with the tsL141 mutant polymerase. The position of the tsL141 mutation (737) and that of a second site mutation (771) that suppresses its phenotype are C-terminal to the regions where T4 polymerase shares homology with eukaryotic DNA polymerases, and may be in a region important for its interactions with the other T4 replication 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：这些蛋白质包括T4 DNA 聚合酶（基因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 引物解旋酶、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。 使用其中可光活化的芳基叠氮化物可逐步移动的系统 通过引物的30个碱基区域，我们发现， 蛋白和32蛋白可以在存在下交联到引物上 一种不可水解的ATP类似物 三人的初始绑定 辅助蛋白和ATP的32蛋白覆盖的引物模板， 接着是ATP水解，聚合酶的结合，以及每个 辅助蛋白质，以产生能够加工DNA的复合物 合成. 链置换合成所需的蛋白质间相互作用。 T4 DNA聚合酶的突变可以降低其相互作用的能力， 与允许链置换合成的蛋白质 型酶。 纯化的tsL 141突变体聚合酶（丙氨酸变为丙氨酸）被纯化。 737处的缬氨酸）被辅助蛋白和32蛋白刺激，但 在单链模板上的暂停位点被捕获的频率更高 野生型，几乎不进行链置换， 在叉状DNA模板上与辅助蛋白，32蛋白， 和41蛋白解旋酶。 我们最近克隆并纯化了 T4基因59蛋白的同源性，这增加了DNA解旋的41 解旋酶 我们发现添加这种59蛋白是绝对必要的 用于用tsL 141突变聚合酶进行链置换合成。 的 tsL 141突变的位置（737）和第二位点突变的位置 (771)抑制其表型的基因位于T4 聚合酶与真核DNA聚合酶具有同源性，并且可以在一个特定的基因组中。 与其他T4复制相互作用的重要区域 proteins.