ENZYMATIC MECHANISMS OF DNA REPLICATION--THE BACTERIOPHAGE T4 SYSTEM

DNA复制的酶促机制--噬菌体T4系统

• 批准号: 3917571
• 负责人: N G NOSSAL
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3917571
• 关键词: DNA binding protein; DNA directed DNA polymerase; Escherichia coli; bacterial genetics; bacteriophage T4; binding proteins; chemical chain length; cofactor; enzyme complex; enzyme induction /repression; enzyme mechanism; gene expression; microorganism genetics; nucleic acid sequence; nucleotide metabolism; ribonuclease III; virus genetics; virus protein

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.

在急诊双链DNA的大肠杆菌噬菌体T4模型系统 复制，在体外实现有效的DNA复制， T4噬菌体编码的7种纯化蛋白质：T4 DNA聚合酶 (gene 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， 41和61蛋白质，它们一起作为引发酶和 解旋酶 聚合酶-辅助蛋白相互作用。 三个配件 蛋白质的功能就像一个滑动钳， 到引物模板。 聚合酶和44/62蛋白结合 在不存在下， ATP 45蛋白的后续结合需要ATP以及两者 聚合酶和44/62蛋白复合物。 叉状模板上的链置换合成。 一个50的基地 合成叉状DNA底物一侧的区域 与滞后链模板相对应， 前导链置换合成的快速相互作用， 表明前导链聚合酶和/或其辅助酶 蛋白质与两条模板链在 复制分叉。 预成型的叉子也允许41蛋白- 依赖链置换合成中的一些分子 32蛋白的缺失 这些结果与一个模型一致 其中41蛋白解旋酶控制双链体的解旋， 而32蛋白则起到防止链再退火的作用 解开了 引物解旋酶与其他复制蛋白的相互作用。 一 41蛋白的胰蛋白酶片段，缺失17或20个氨基酸， C-末端，保留了引物和解旋酶的活性， 完整的酶，但不能催化这些活动， 其他复制蛋白的存在。 引物合成 61蛋白，以及完整的41蛋白或胰蛋白酶片段， 被高浓度的32蛋白抑制。 聚合酶 辅助蛋白逆转这种抑制与完整的41蛋白 但不是41号碎片 因此，C-末端的改变 41个蛋白质中的一个阻止了与辅助蛋白质的相互作用 这是在32蛋白包被的DNA上合成引物所必需的。