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: 3875724
• 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/3875724
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA primase; DNA replication; Escherichia coli; RNA biosynthesis; bacterial genetics; bacteriophage T4; chemical chain length; complementary DNA; enzyme complex; enzyme induction /repression; enzyme mechanism; eukaryote; exonuclease; gene expression; gene rearrangement; genome; ligase; microorganism genetics; molecular cloning; nucleic acid sequence; pancreatic ribonuclease; protein sequence; ribonuclease III; site directed mutagenesis; virus genetics; virus protein

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 and 61 primase-helicase, RNase H, and DNA ligase. We have used DNA primers bearing photoactivated crosslinking residues to label 5 proteins of the T4 DNA replication complex: polymerase, the 44/62 and 45 polymerase accessory proteins, and 32 single-stranded DNA binding protein. We have determined the relative positions of each of the 5 proteins and the length of DNA in contact with the proteins. We find that the polymerase is bound at the primer terminus and covers 5-7 bases of duplex DNA. Efficient labeling of the polymerase requires all 5 proteins and ATP. When the crosslinkable residue is 14-20 bases from the 31 end of the primer-terminus, the 45 accessory protein is labeled, but only,in the presence of all 5 proteins and ATP. When the primer-terminus is 25 bases from the crosslinkable residue, labeling of the 45 protein is greatly decreased. Our data suggest that the 44/62 complex resides between the polymerase and the 45 proteins. Identification of a T4 Gene Encoding the RNase H Which Removes RNA Primers on the Lagging Strand. We have cloned the T4 DNA encoding an open reading frame which matches the N-terminal sequence of an RNase H which we previously purified from T4 infected cells. Plasmids containing this gene, located upstream of gene 33, express a protein whose size and RNase H and 5' to 3' DNA exonuclease activities are identical to those of the phage-encoded enzyme. This 35 kd protein has three regions with significant amino acid sequence similarity to those of the 5' to 3' exonuclease domain of E. coli pol I and the T7 gene 6 exonuclease. Essential Regions of T4 DNA Polymerase. T4 DNA polymerase has six regions which share sequence similarity with eukaryotic DNA polymerases. To determine the function of shared and unique regions, we are characterizing altered T4 DNA polymerases produced by site-specific mutagenesis of the cloned gene.

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复制是 用T4噬菌体编码的纯化蛋白质实现：T4 DNA聚合酶 (gene 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和61引物解旋酶， RNase H和DNA连接酶。 我们已经使用带有光活化交联残基的DNA引物， 标记T4 DNA复制复合物的5种蛋白质：聚合酶，44/62 45种聚合酶辅助蛋白和32种单链DNA结合蛋白 蛋白我们已经确定了这5个物体的相对位置 蛋白质和与蛋白质接触的DNA的长度。我们发现 聚合酶结合在引物末端并覆盖5-7个碱基 双链DNA聚合酶的有效标记需要所有5种蛋白质 和ATP。当可交联残基是从31端起的14-20个碱基时， 在引物末端，45辅助蛋白被标记，但仅在 所有5种蛋白质和ATP。当引物末端为25个碱基时 从可交联的残基中，45蛋白的标记大大降低了 降低我们的数据表明，44/62复合体位于 聚合酶和45种蛋白质。 一个编码RNA酶H的T4基因的鉴定 在落后海岸我们克隆了编码开放阅读的T4 DNA 框架，该框架与RNase H的N-末端序列相匹配， 之前从T4感染的细胞中纯化。含有该基因的质粒， 位于基因33的上游，表达大小和RNA酶H的蛋白质， 5'至3' DNA外切核酸酶活性与那些具有5 '至3' DNA外切核酸酶活性的人相同。 噬菌体编码的酶。该35 kd蛋白具有三个区域， 与5'至3'核酸外切酶结构域的氨基酸序列相似性 大肠coli pol I和T7基因6外切核酸酶。 T4 DNA聚合酶的关键区域T4 DNA聚合酶有六个区域 其与真核DNA聚合酶共享序列相似性。到 确定共享和独特区域的功能，我们正在表征 改变的T4 DNA聚合酶产生的位点特异性诱变的 克隆基因