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: 4689435
• 负责人: N G NOSSAL
• 金额: --
• 依托单位: NAT INST OF ARTHRITIS, DIABETES, DIGESTIVE & 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/4689435
• 关键词: DNA directed DNA polymerase; Escherichia coli; bacterial genetics; bacteriophage T4; chemical chain length; circular DNA; electron microscopy; endonuclease; enzyme induction /repression; microorganism genetics; molecular cloning; nuclease; nucleic acid metabolism; temperature sensitive mutant

We are studying the E. coli bacteriophage T4 as a model system for duplex DNA replication. Efficient DNA synthesis in vitro can be achieved with a mixture of 7 purified proteins encoded by T4 phage, which include T4 DNA polymerase (gene 43 product), the gene 32 DNA helix-destabilizing protein, the gene 44/62 and gene 45 polymerase accessory proteins, and the gene 41 and gene 61 priming proteins. We have cloned the genes for the T4 61 and 41 primase proteins and designed new procedures for the large-scale purification of each protein. At very high concentrations, 61 protein alone catalyzes some oligonucleotide synthesis on single-stranded DNA templates, yielding mainly dinucleotides (pppAC) in contrast to the pentanucleotides formed by the combined action of the 61 and 41 primase proteins. Oligonucleotides made without 41 protein prime DNA synthesis by T4 polymerase and its accessory proteins, but both primer and DNA synthesis are greatly stimulated by the 41 protein. The DNA unwinding activity (helicase) of 41 protein is increased more than 30-fold by 61 protein, which has no independent unwinding activity. We are screening mutant forms of 41 protein, produced by existing mutants and by in vitro mutagenesis of the cloned gene, to identify regions of 41 protein required for its primase, helicase, and DNA-dependent nucleotidase activities. Plasmids encoding the T4 uvsX recombination protein decrease the UV sensitivity of T4 mutants in the uvsX gene. Purified uvsX protein catalyzes ATP hydrolysis to ADP and AMP and strand exchange between homologous single-stranded circular and linear duplex DNA. Strand exchange is stimulated by T4 gene 32 protein and by a 3' single-stranded extension on the duplex DNA. We are using the plasmids containing the T4 uvsX and 41 helicase genes and those containing the 61 primase and DNA adenine methylase genes to identify sequences and factors regulating the expression of the T4 DNA in these two neighboring regions.

我们正在研究E。大肠杆菌噬菌体T4作为双链体的模型系统 DNA复制。 体外有效的DNA合成可以通过以下方法实现： 由T4噬菌体编码的7种纯化蛋白质的混合物，其中包括T4 DNA 聚合酶（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，以及基因41 和基因61引发蛋白。 我们已经克隆了T4 61和41引发酶蛋白的基因，并设计了 大规模纯化每种蛋白质的新程序。 以非常 高浓度61蛋白单独催化一些寡核苷酸 在单链DNA模板上合成，主要产生二核苷酸 （pppAC）与通过组合作用形成的五核苷酸相反， 61和41引发酶蛋白。 在没有41的情况下制备的寡核苷酸 蛋白质引发T4聚合酶及其辅助蛋白的DNA合成， 但引物和DNA合成都受到41 蛋白 41蛋白的DNA解旋活性（解旋酶）增加 超过30倍的61蛋白，它没有独立的解旋 活动 我们正在筛选41蛋白的突变形式， 现有的突变体，并通过体外诱变克隆的基因， 鉴定41种蛋白质的引发酶、解旋酶和 DNA依赖性核苷酸酶活性。 编码T4 uvsX重组蛋白的质粒降低了UV uvsX基因中T4突变体的敏感性。 纯化的uvsX蛋白 催化ATP水解为ADP和AMP， 同源单链环状和线性双链体DNA。 链交换 受T4基因32蛋白和3'单链延伸刺激 双链体DNA上 我们使用含有T4 uvsX和41解旋酶基因的质粒， 那些含有61个引物酶和DNA腺嘌呤甲基化酶基因，以确定 序列和因子调节T4 DNA在这两种细胞中的表达， 邻近地区。