ENZYMATIC MECHANISMS OF DNA REPLICATION--THE BACTERIOPHAGE T4 SYSTEM

我们正在研究大肠杆菌噬菌体T4作为双链的模型系统 DNA复制。高效的体外DNA合成可以通过一种 T4噬菌体编码的7种纯化蛋白的混合物，其中包括T4DNA 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启动蛋白。 我们已经克隆了T461和41Primase蛋白的基因，并设计了 大规模提纯每种蛋白质的新程序。在非常 高浓度，61蛋白单独催化一些寡核苷酸 以二核苷酸为主的单链DNA模板的合成 (PppAC)，而不是由联合作用形成的五核苷酸 在61和41个启动酶蛋白中。不含41的寡核苷酸 T4聚合酶及其辅助蛋白合成DNA的蛋白质， 但是，41对引物和DNA合成都有很大的刺激作用 蛋白。41蛋白的DNA解旋活性(解旋酶)增加 超过30倍的61个蛋白质，没有独立的解离 活动。我们正在筛选41种蛋白质的突变形式，这些蛋白质由 现有的突变体和通过体外诱变克隆的基因，以 确定其启动酶、解旋酶和蛋白质所需的41个蛋白质区域 依赖DNA的核苷酸酶活性。 编码T4 uvsX重组蛋白的质粒会降低紫外线 UvsX基因T4突变体的敏感性。纯化的uvsX蛋白 催化ATP水解为ADP和AMP，并在 同源单链环状和线状双链DNA。链交换 是由T4基因32蛋白和3‘单链延伸刺激的 在双链DNA上。 我们使用的是含有T4 uvsX和41解旋酶基因的质粒 对那些含有61个底物酶和DNA腺嘌呤甲基酶基因的人进行鉴定 T4DNA在这两种细胞中表达的序列和调控因素 邻近地区。

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.