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（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。我们正在与 Tim Meuser和克雷格海德，NIAMS，确定T4的结构 X射线衍射法测定DNA复制蛋白质。我们最近的工作是 旨在确定T4 RNA酶H如何协调引物去除 与滞后链和前导链合成中的其他步骤一起， 是什么控制了多少DNA随着RNA引物被沿着去除，以及 阐明了核酸酶的结构和水解机理。 T4 RNaseH是一种5'至3'核酸外切酶，其从RNA聚合物中去除RNA引物。 DNA复制叉的滞后链，是RAD 2的成员 真核和原核复制和修复核酸酶家族。 在晶体结构中，最保守的酸性残基是 聚在一起围绕两个镁在裂缝足够宽， DNA.定点诱变表明，两个Mg中只有一个是 催化作用所必需的，而裂缝上方的无序区域 与DNA结合有关T4基因32蛋白将T4 RNA酶H转化为 进行性核酸外切酶，但抑制其瓣核酸内切酶，表明 32蛋白在控制T4 RNase H降解中的重要作用 RNA引物和相邻DNA在每个滞后链循环。 我们 正在研究基因59蛋白刺激DNA的机制， 解旋的41解旋酶，和引物合成依赖于这两个 41和61蛋白质。解旋酶59的晶体结构解析 蛋白质组装正在进行中。

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. We are collaborating with Tim Meuser and Craig Hyde, NIAMS, to determine the structure of the T4 DNA replication proteins by X-ray diffraction. Our recent work has been directed at determining how primer removal by T4 RNase H is coordinated with other steps in lagging and leading strand synthesis, understanding what controls how much DNA is removed along with the RNA primer, and elucidating the structure and hydrolytic mechanism of the nuclease. T4 RNaseH is a 5' to 3' exonuclease that removes RNA primers from the lagging strand of the DNA replication fork, and is a member of the RAD2 family of eukaryotic and prokaryotic replication and repair nucleases. In the crystal structure, the most conserved acidic residues are clustered together surrounding two Mg in a cleft wide enough for single DNA. Site-directed mutagenesis indicates that only one of the two Mg is essential for catalysis, and that the disordered region above the cleft is involved in DNA binding. T4 gene 32 protein converts T4 RNase H into a processive exonuclease, but inhibits its flap endonuclease, suggesting an important role for 32 protein in controlling T4 RNase H degradation of RNA primers and adjacent DNA during each lagging strand cycle. 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. Solution of the crystal structure of the 59 helicase assembly protein is in progress.