ENZYMATIC MECHANISMS OF DNA REPLICATION--THE BACTERIOPHAGE T4 SYSTEM

我们正在继续研究E.Coli噬菌体T4模型系统 用于在体外高效复制DNA的双链DNA复制 是通过T4噬菌体：T4DNA聚合酶编码的纯化蛋白实现的 (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启动酶- 解旋酶、核糖核酸酶H和DNA连接酶。 T4DNA复制蛋白的结构。我们正在与 Tim Meuser和Craig Hyde，NIAMS，以确定每个 用单晶X射线衍射法测定T4DNA复制蛋白。 T4 RNAseH是一种5‘到3’的RNA：DNA和DNA：DNA核酸酶，其结构具有 已被重原子MIR分解为2.8埃的正交晶系 水晶形状。更高分辨率的单斜晶型随后被解为 使用精化模型进行分子置换。差值电子 BaCl2取代MgCl2的正交晶型密度图 清楚地显示在假定的活性部位中有一个单一的钡结合部位 被最高含量的氨基酸所包围 在与T4相关的原核和真核核酸酶中保守 RNASEH。我们正在构建单点突变来测试我们的模型 产生一种失活的酶，使其与其双链底物结晶。二 基因59解旋酶组装蛋白的正交晶型 衍射到高分辨率。T4启动子酶的功能和结构- 解旋酶复合体。我们正在研究基因59 蛋白质通过41解旋酶刺激DNA解链，并合成引物 依赖于41和61蛋白，使用亲和层析， 凝胶过滤和化学交联剂。ATP增加了交叉- 41和59蛋白与可光激活DNA的连接 底物，不能被三磷酸腺苷取代；59和41个蛋白质 与DNA的交叉连接在更大程度上存在于 其他蛋白质。T4DNA 737-772区的结构与功能 聚合酶。我们的结构分析表明，737-772区域， 以前被证明对聚合酶的保真度有影响，可能 代表一对在G753处弯曲并相互作用的螺旋 通过疏水相互作用在分子内相互作用，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对这些基因的预测 模特们。

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.