ENZYMATIC MECHANISMS OF DNA REPLICATION--THE BACTERIOPHAGE T4 SYSTEM

在双链DNA的大肠杆菌噬菌体T4模型系统中 复制，高效的体外DNA复制是通过 T4噬菌体：T4DNA聚合酶编码的7种纯化蛋白 (基因43产物)，基因32 DNA螺旋不稳定蛋白， 基因44/62和基因45聚合酶辅助蛋白及其基因 41和61蛋白，它们共同发挥着启动酶和 解旋酶。 聚合酶-辅助蛋白相互作用。三个附属品 蛋白质起着滑动钳子的作用，以保持聚合酶的结合 添加到底物模板。聚合酶与44/62蛋白结合 不依赖于寡核苷酸启动的单链DNA 三磷酸腺苷。45蛋白的后续结合需要三磷酸腺苷和 聚合酶和44/62蛋白复合体。 叉形模板上的链置换合成。50个基数 人工合成的分叉DNA底物侧边的区域 对应于滞后链模板允许同步和 主链置换合成的快速相互作用， 表明前导链聚合酶和/或其附件 蛋白质在两个模板链上都有接触 复制分叉。预制的叉子还允许41种蛋白质- 依赖链置换合成法在某些分子上的应用 缺少32个蛋白质。这些结果与一个模型一致 其中41蛋白解旋酶控制双链的解离， 而32蛋白的作用是防止链的再退火。 放松。 Primase-Helicase与其他复制蛋白的相互作用。一个 41个蛋白质的胰酶片段，缺失17或20个氨基酸 在C末端，保留了底物酶和解旋酶的活性。 完整的酶，但不能催化这些活性在 其他复制蛋白的存在。底物合成方法 61蛋白，以及完整的41蛋白或胰酶片段， 被高浓度的32蛋白抑制。聚合酶 辅助蛋白与完整的41蛋白一起逆转这种抑制作用 但与41片段不同。因此，C-末端的改变 阻止了与辅助蛋白的相互作用 这是在32蛋白包被DNA上合成引物所必需的。

In the E. coli bacteriophage T4 model system for duplex DNA replication, efficient DNA replication in vitro is achieved with seven purified proteins encoded by T4 phage: T4 DNA polymerase (gene 43 product), gene 32 DNA helix-destabilizing protein, the gene 44/62 and gene 45 polymerase accessory proteins, and the genes 41 and 61 proteins, which together function as a primase and a helicase. Polymerase-Accessory Protein Interactions. The three accessory proteins function as a sliding clamp to keep the polymerase bound to the primer-template. Polymerase and the 44/62 protein bind independently to oligonucleotide-primed ssDNA in the absence of ATP. Subsequent binding of 45 protein requires ATP as well as both polymerase and the 44/62 protein complex. Strand Displacement Synthesis on Forked Templates. A 50 base region on the side of a synthetic forked DNA substrate corresponding to the lagging strand template allows synchronous and rapid interaction of leading strand displacement synthesis, indicating that the leading strand polymerase and/or its accessory proteins is in contact with both template strands at the replication fork. The preformed fork also permits 41 protein- dependent strand displacement synthesis on some molecules in the absence of 32 protein. These results are consistent with a model in which 41 protein helicase controls unwinding of the duplex, while 32 protein acts to prevent reannealing of the strands unwound. Primase-Helicase Interactions with Other Replication Proteins. A tryptic fragment of 41 protein, missing 17 or 20 amino acids from the C-terminus, retains the primase and helicase activities of the intact enzyme, but is unable to catalyze these activities in the presence of the other replication proteins. Primer synthesis by 61 protein, and either intact 41 protein or the tryptic fragment, is inhibited by high concentrations of 32 protein. The polymerase accessory proteins reverse this inhibition with intact 41 protein but not with the 41 fragment. Thus, alteration of the C-terminus of 41 protein prevents an interaction with the accessory proteins which is necessary for primer synthesis on 32 protein coated DNA.