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噬菌体编码的纯化蛋白获得：包括T4DNA 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 Primase-Helicase、RNaseH和DNA连接酶。 聚合酶和辅助蛋白在引物模板上的组装。 使用其中可光激活的芳基叠氮化物可以逐步移动的系统 通过该引物的30个碱基区，我们发现该附件 在存在的情况下，可以将蛋白质和32蛋白质交联到该引物上 一种非水解性的三磷酸腺苷类似物。三者的最初约束力 辅助蛋白质和三磷酸腺苷到32个蛋白质覆盖的引物模板是 然后是ATP的水解，聚合酶的结合，以及每一个 产生能够进行DNA的复合体的辅助蛋白质 综合。 链置换合成所需的蛋白质-蛋白质相互作用。 T4DNA聚合酶的突变会降低其相互作用的能力 与允许与野生动物进行链置换合成的蛋白质 键入酶。纯化的tsL14L突变聚合酶(丙氨酸变为 Valine at 737)受到辅助蛋白和32蛋白的刺激，但 在单链模板上的停顿位置更频繁地被捕获 野生型，并且几乎不进行链移位 在带有辅助蛋白32蛋白的分叉DNA模板上合成 和41蛋白解旋酶。我们最近克隆并纯化了 同质性的T4基因59蛋白，使DNA解离增加了41% 解旋酶。我们发现添加这种59蛋白是绝对必要的 用于与tsL141突变聚合酶进行链置换合成。这个 TsL141突变(737)的位置和第二个点突变的位置 抑制其表型的(771)是C-末端到T4的区域 聚合酶与真核DNA聚合酶有同源性，可能在 对其与其他T4复制相互作用很重要的区域 蛋白质。

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: these include 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. Using a system in which a photoactivatable aryl azide can be moved stepwise through a thirty base region of the primer, we find that the accessory proteins and 32 protein can be cross-linked to the primer in the presence of a non-hydrolyzable ATP analogue. The initial binding of the three accessory proteins and ATP to a 32 protein-covered primer-template is followed by ATP hydrolysis, binding of polymerase, and movement of each of the accessory proteins to yield a complex capable of processive DNA synthesis. Protein-protein interactions required for strand displacement synthesis. Mutations in the T4 DNA polymerase can decrease its ability to interact with the proteins that allow strand displacement synthesis with the wild type enzyme. The purified tsLl4l mutant polymerase (alanine changed to valine at 737) is stimulated by the accessory proteins and 32 protein, but is arrested at pause sites on single-stranded templates more frequently that the wild type, and carries out practically no strand displacement synthesis on a forked DNA template with the accessory proteins, 32 protein, and the 41 protein helicase. We have recently cloned and purified to homogeneity the T4 gene 59 protein, which increases DNA unwinding by the 41 helicase. We find that addition of this 59 protein is absolutely required for strand displacement synthesis with the tsL141 mutant polymerase. The position of the tsL141 mutation (737) and that of a second site mutation (771) that suppresses its phenotype are C-terminal to the regions where T4 polymerase shares homology with eukaryotic DNA polymerases, and may be in a region important for its interactions with the other T4 replication proteins.