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: 3776950
• 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/3776950
• 关键词: DNA directed DNA polymerase; DNA primase; DNA replication; X ray crystallography; bacteriophage T4; enzyme mechanism; exonuclease; genetic regulation; helicase; intermolecular interaction; ligase; molecular genetics; mutant; protein structure; ribonuclease III; virus DNA; virus genetics

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. Mutations in T4 DNA polymerase. Our studies of an antimutator mutant in T4 DNA polymerase (A737V) indicate that the accuracy of the polymerase can be altered by changing the rate at which the growing strand of the duplex moves between the polymerase and exonuclease active sites on the enzyme. This single amino acid substitution decreases the processivity of the polymerase activity and increases the processivity of the proofreading exonuclease activity. These processivity changes are reversed by the compensating L771F mutation. Interactions between replication proteins. We are studying how interactions between proteins in the replication complex regulate synthesis on the leading and lagging strands. DNA templates with photo-activatable cross-linking residues in the fork ahead of the primer have been constructed, and are being used to determine whether any of the accessory proteins are in front of polymerase, and to learn how and where the 61, 41, and 59 protein primase-helicase components assemble at the fork. In studies of the mechanism by which the gene 59 protein stimulates the 41/61 protein primase-helicase, we have demonstrated a physical interaction between the 59 and 41 proteins in the absence of DNA. T4 RNaseH. In a nonpermissive (RNaseH defective) host, a T4 mutant with a deletion in the phage RNaseH gene makes few viable progeny, and accumulates short DNA chains characteristic of unligated lagging strand fragments. We are using purified T4 RNaseH to study how primer removal and gap filling are coordinated. Structure of the T4 replication proteins. We are collaborating 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 用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、RNA酶H和DNA连接酶。 T4 DNA突变 聚合酶。 T4 DNA聚合酶抗突变体的研究 （A737 V）表明聚合酶的准确性可以通过以下改变： 改变双链体的生长链移动的速率 在酶上的聚合酶和核酸外切酶活性位点之间。 这 单个氨基酸取代降低了细胞的持续合成能力， 聚合酶的活性，并增加了校对的持续性 外切核酸酶活性。 这些持续合成能力的变化被 补偿L771 F突变。 复制蛋白之间的相互作用。 我们正在研究复制过程中蛋白质之间的相互作用 复合物调节前导链和滞后链的合成。 DNA 在叉中具有可光活化的交联残基的模板 在引物之前已经构建，并用于 确定是否有任何辅助蛋白在 聚合酶，并了解61，41和59蛋白如何以及在哪里 引物-解旋酶组分在叉处组装。 在研究 基因59蛋白刺激41/61蛋白的机制 引物解旋酶，我们已经证明了物理相互作用之间 59和41蛋白在没有DNA的情况下。 T4 RNA酶H。中 非允许性（RNaseH缺陷型）宿主，一种T4突变体， 噬菌体RNaseH基因很少产生有活力的后代， 未连接的滞后链片段的DNA链特征。 我们 使用纯化的T4 RNaseH研究引物去除和缺口填充是如何 协调一致。 T4复制蛋白的结构。 我们正在与 克雷格海德，NIAMS，试图结晶这些蛋白质。