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: 5202049
• 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/5202049
• 关键词: DNA directed DNA polymerase; DNA primase; DNA replication; X ray crystallography; active sites; affinity chromatography; bacteriophage T4; crosslink; crystallization; enzyme complex; enzyme mechanism; enzyme structure; gel filtration chromatography; helicase; hydropathy; intermolecular interaction; ribonuclease III; site directed mutagenesis; 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. The structure of T4 RNaseH, a 5' to 3' RNA:DNA and DNA:DNA nuclease, has been solved by heavy atom MIR to 2.8 angstroms for an orthorhombic crystal form. A monoclinic form of higher resolution was then solved by molecular replacement using the refined model. The difference electron density map of the orthorhombic crystal form with BaCl2 replacing MgCl2 clearly shows a single barium binding site in the putative active site of the enzyme, surrounded by the amino acids that are most highly conserved in the prokaryotic and eukaryotic nucleases related to T4 RNaseH. We are constructing single site mutations to test our model and produce an inactive enzyme to crystalize with its duplex substrates. Two orthorhombic crystal forms of the gene 59 helicase assembly protein diffract to high resolution. Function and structure of the T4 primase- helicase complex. We are studying the mechanism by which the gene 59 protein stimulates DNA unwinding by the 41 helicase, and primer synthesis dependent on both the 41 and 61 proteins, using affinity chromatography, gel filtration, and chemical cross-linking. ATP increases the cross- linking of both the 41 and 59 proteins to photoactivatable DNA substrates, and cannot be replaced by ATP'S; 59 and 41 proteins each cross-link to the DNA to a much greater extent in the presence of the other protein. Structure and function of the 737-772 region of T4 DNA polymerase. Our structural analysis suggests that the 737-772 region, previously been shown to have effects on polymerase fidelity, may represent a pair of helices bent at G753, and interacting intramolecularly with one another via hydrophobic interactions, or alternatively, could be a dimerization domain between two polymerases. Site-direct mutagenesis is being used to test predictions of these models.

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 (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。 T4 DNA复制蛋白的结构。 我们正在与 Tim Meuser和克雷格海德，NIAMS，以确定每一个的结构， T4 DNA复制蛋白的单晶X射线衍射分析。 T4 RNaseH是一种5'至3' RNA：DNA和DNA：DNA核酸酶，其结构具有 通过重原子MIR解决了正交晶系的2.8埃 晶体形式。 一个更高分辨率的单斜形式，然后解决， 分子置换使用改进的模型。 差电子 BaCl 2替代MgCl 2的正交晶型的密度图 清楚地显示了一个单一的钡结合位点在推定的活性位点 被最高浓度的氨基酸所包围 T4相关的原核和真核核酸酶中保守 RNA酶H。 我们正在构建单位点突变来测试我们的模型， 产生一种失活的酶与其双链底物一起结晶。 两 基因59解旋酶组装蛋白的正交晶型 以高分辨率显示。 T4引发酶的功能和结构 解旋酶复合物 我们正在研究基因59 蛋白质通过41解旋酶刺激DNA解旋和引物合成 依赖于41和61蛋白，使用亲和层析， 凝胶过滤和化学交联。 ATP增加了交叉- 将41和59蛋白质连接到光活化DNA 底物，不能被ATP取代; 59和41蛋白质各 在存在的情况下，与DNA的交联程度要大得多 其他蛋白质T4 DNA 737-772区的结构和功能 聚合酶。 我们的结构分析表明737-772区域， 先前已显示对聚合酶保真度有影响， 表示在G753处弯曲的一对螺旋， 通过疏水相互作用彼此分子内结合，或 或者可以是两个聚合酶之间的二聚化结构域。 定点突变被用来测试这些预测 模型