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: 3754867
• 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/3754867
• 关键词: DNA directed DNA polymerase; DNA primase; DNA replication; X ray crystallography; affinity chromatography; bacteriophage T4; crosslink; enzyme mechanism; exonuclease; genetic regulation; helicase; hydrolysis; intermolecular interaction; ligase; molecular genetics; protein structure function; ribonuclease III; virus DNA; virus genetics; virus replication

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. Structure of the T4 DNA replication proteins. We are collaborating with Tim Meuser and Craig Hyde, NIAMS, to determine the structure of each of the T4 DNA replication proteins using single crystal X-ray diffraction. We have obtained high resolution data (2.5 angstroms) on large single crystals of RNaseH and the gene 59 helicase assembly protein, and are screening heavy atom derivatives for phasing potential. Assembly of replication proteins on a forked DNA template. We have constructed substrates with a photoactivatable residue in the fork ahead of a primer to learn how and where the polymerase and polymerase accessory proteins and the gene 61, 41, and 59 protein primase-helicase components assemble at the fork. ATP hydrolysis by the 44/62 complex is required to assemble the 45 protein on a primer in a form in which it can move forward on the single-stranded DNA, as shown by its cross-linking to the fork ahead. Polymerase adds to this complex, occupying a position ahead of the 45 protein, and is cross-linked only when there is no gap between the primer and the fork. Function and structure of the T4 primase-helicase complex. We are studying the mechanism by which the gene 59 proteins stimulates DNA unwinding by the 41 helicase, and primer synthesis dependent on both the 41 and 61 proteins. We are probing the physical interactions between these proteins by affinity chromatography, centrifugation, and chemical cross- linking. T4 RNaseH. We are using purified T4 RNaseH to determine how primer removal by RNaseH and gap filling by polymerase are coordinated on the lagging strand. During 5' to 3' hydrolysis T4 RNaseH removes oligonucleotides of 1 to 5b. The rate of digestion and length of the products are increased by 32 protein. T4 DNA polymerase synthesis from an upstream fragment stimulates RNaseH hydrolysis on the adjacent fragment.

我们正在继续研究大肠杆菌噬菌体 T4 模型系统 用于双链 DNA 复制，其中有效的 DNA 体外复制 通过 T4 噬菌体编码的纯化蛋白质实现：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 引物酶- 解旋酶、RNase H 和 DNA 连接酶。 T4 DNA 复制的结构 蛋白质。 我们正在与 NIAMS 的 Tim Meuser 和 Craig Hyde 合作， 确定每个 T4 DNA 复制蛋白的结构 使用单晶X射线衍射。 我们已经获得了高分辨率 RNaseH 和基因 59 的大单晶数据（2.5 埃） 解旋酶组装蛋白，并正在筛选重原子衍生物 分相潜力。 复制蛋白在分叉 DNA 上的组装 模板。 我们构建了带有光活化残留物的基材 在底漆之前的叉子中，了解聚合酶和聚合酶的方式和位置 聚合酶辅助蛋白和基因 61、41 和 59 蛋白 引物酶-解旋酶成分在叉处组装。 ATP 水解 需要 44/62 复合体才能在引物上组装 45 蛋白 它可以在单链DNA上向前移动的形式，如图所示 通过其与前面的叉子的交联。 聚合酶添加到该复合物中， 占据45蛋白前面的位置，并且仅是交联的 当底漆和前叉之间没有间隙时。 功能和 T4引物酶-解旋酶复合物的结构。 我们正在研究 基因 59 蛋白刺激 DNA 解旋的机制 41 解旋酶，以及依赖于 41 和 61 的引物合成 蛋白质。 我们正在探索这些之间的物理相互作用 通过亲和层析、离心和化学交叉法分离蛋白质 链接。 T4 RNaseH。 我们使用纯化的 T4 RNaseH 来确定 RNaseH 的引物去除和聚合酶的间隙填充相协调 在滞后链上。 在 5' 至 3' 水解过程中，T4 RNaseH 去除 1至5b的寡核苷酸。 消化速度和长度 产品增加了32种蛋白质。 T4 DNA 聚合酶合成 上游片段刺激邻近的 RNaseH 水解 分段。