ENZYMATIC MECHANISMS OF DNA REPLICATION--THE BACTERIOPHAGE T4 SYSTEM

我们正在继续我们对大肠杆菌噬菌体T4模型系统的研究 用于其中高效的体外DNA复制的双链DNA复制 用T4噬菌体：T4DNA聚合酶编码的纯化蛋白获得 (基因43)、基因32 DNA螺旋不稳定蛋白、基因44/62和基因 45个聚合酶辅助蛋白，基因41、61和59。 解旋酶、核糖核酸酶H和DNA连接酶。 聚合酶和辅助蛋白在引物模板上的组装。 我们之前已经利用蛋白质的交联性来实现光激活 引物中特定核苷酸上的残留物确定其位置 聚合酶和引物上的每一种辅助蛋白。为了 将这些蛋白质定位在模板链上，并研究 通过凝胶迁移率改变分析组装络合物，我们是 进行系统分析以确定最小尺寸的底物- 模板将允许综合体的适当组装。 T4DNA聚合酶基因突变的构建及分析。T4 DNA 聚合酶与一个大家族具有广泛的氨基酸序列相似性 原核和真核DNA聚合酶。我们在试管中使用 突变以确定这些保守区的功能并尝试 确定聚合酶与其相互作用的重要区域 其他T4复制蛋白。我们已经构建了一个聚合酶 缺乏编辑核酸外切酶通过改变保守的氨基酸在 N-端子区。该突变体保持其聚合酶活性，并 与辅助蛋白正常相互作用，T4噬菌体与 核酸外切酶缺陷聚合酶基因的突变率很高 活着。 T4基因59解旋酶组装因子的功能。增加59个 蛋白质对其他七种复制蛋白质的作用显著增加 双链DNA的合成。我们目前正在研究如何以及在哪里实现这一点 合成开始了。 T4 RNAseH。我们已经鉴定了一个编码RNAseH的T4基因，并表明 纯化后的蛋白可在体外去除RNA引物。我们已经构建了一个 在RNAseH基因内缺失的噬菌体突变体，并表明这 在缺乏RNAseH和5-3的宿主中，基因是必不可少的 Pol I核酸外切酶。 T4复制蛋白的结构。我们已经开始合作了 与NIAMS的克雷格·海德合作，试图使这些蛋白质结晶。

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. Assembly of the polymerase and accessory proteins on the primer template. We have previously used cross-linking of proteins to photo-activatable residues at specific nucleotides in the primer to determine the position of polymerase and each of the accessory proteins on the primer. In order to position these proteins on the template strand, and to study the steps in the assembly of the complex by a gel-mobility shift assay, we are conducting a systematic analysis to determine the minimum size primer- template that will allow the proper assembly of the complex. Construction and analysis of mutations in T4 DNA polymerase. T4 DNA polymerase has extensive amino acid sequence similarity with a large family of prokaryotic and eukaryotic DNA polymerases. We are using in vitro mutagenesis to determine the function of these conserved regions and to try to identify regions of the polymerase important for its interaction with the other T4 replication proteins. We have constructed a polymerase lacking the editing exonuclease by changing a conserved amino acid in the N-terminal region. This mutant retains its polymerase activity and interacts normally with the accessory proteins, T4 phage with the exonuclease defective polymerase gene have a very high rate of mutation in vivo. Function of the T4 gene 59 helicase assembly factor. The addition of 59 protein to the other seven replication proteins gives a marked increase in synthesis on duplex DNA. We are currently studying how and where this synthesis is initiated. T4 RNaseH. We have identified a T4 gene encoding an RNaseH, and shown that the purified protein removes RNA primers in vitro. We have constructed a phage mutant with a deletion within the RNaseH gene, and shown that this gene is essential in a host that is lacking both RNaseH and 5 to 3 exonuclease of Pol I. Structure of the T4 replication proteins. We have begun a collaboration with Craig Hyde, NIAMS, to try to crystallize these proteins.