ENZYMATIC MECHANISMS OF DNA REPLICATION--THE BACTERIOPHAGE T4 SYSTEM

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复制的结构 proteins. 我们正在与蒂姆·默瑟和克雷格·海德合作， 为了确定每个T4 DNA复制蛋白的结构， 使用单晶X射线衍射。 我们获得了高分辨率 RNaseH和基因59的大单晶上的数据（2.5埃） 解旋酶组装蛋白，并筛选重原子衍生物， 定相潜力。 复制蛋白在分叉DNA上的组装 template. 我们已经构建了具有光活化残基的基底， 在引物前面的叉中，以了解聚合酶和 聚合酶辅助蛋白和基因61、41和59蛋白 引物-解旋酶组分在叉处组装。 ATP水解 需要44/62复合物在引物上组装45蛋白， 它可以在单链DNA上向前移动，如图所示 通过它的交叉连接到前面的分叉。 聚合酶添加到这个复合物中， 占据45蛋白前面的位置，并且仅交联 当底火和叉子之间没有间隙时。 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解旋酶的引物合成。 proteins. 我们正在探索这些物质之间的物理相互作用 蛋白质通过亲和层析、离心和化学交叉， 链接。 T4 RNA酶H。 我们使用纯化的T4 RNaseH来确定 RNA酶H的引物去除和聚合酶的缺口填充是协调的 在落后的河岸上 在5 '至3'水解期间，T4 RNaseH去除 1至5b的寡核苷酸。 消化率和长度 产品增加了32种蛋白质。 T4 DNA聚合酶合成 一个上游片段刺激RNaseH在邻近的 碎片

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.