Tabor S, Huber H E, Richardson C C
Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115.
J Biol Chem. 1987 Nov 25;262(33):16212-23.
Bacteriophage T7 gene 5 protein has been purified to apparent homogeneity from cells overexpressing its gene several hundred-fold. Gene 5 protein is a DNA polymerase with low processivity; it dissociates from the primer-template after catalyzing the incorporation of 1-50 nucleotides, depending on the salt concentration. Escherichia coli thioredoxin, a host protein that is tightly associated with the gene 5 protein in phage-infected cells, is not required for this activity. Thioredoxin acts as an accessory protein to bestow processivity on the polymerizing reaction; DNA synthesis catalyzed by the gene 5 protein-thioredoxin complex on a single-stranded DNA template can polymerize thousands of nucleotides without dissociation. Conditions that increase the stability of secondary structures in the template (i.e., low temperature or high ionic strength) decrease the processivity. E. coli single-stranded DNA-binding protein stimulates both the rate of elongation and the processivity of the gene 5 protein-thioredoxin complex.
噬菌体T7基因5蛋白已从其基因过表达数百倍的细胞中纯化至表观均一。基因5蛋白是一种持续合成能力较低的DNA聚合酶；根据盐浓度，它在催化1 - 50个核苷酸掺入后会从引物-模板上解离。大肠杆菌硫氧还蛋白是噬菌体感染细胞中与基因5蛋白紧密结合的一种宿主蛋白，该活性并不需要它。硫氧还蛋白作为一种辅助蛋白赋予聚合反应持续合成能力；基因5蛋白-硫氧还蛋白复合物在单链DNA模板上催化的DNA合成能够聚合数千个核苷酸而不解离。增加模板二级结构稳定性的条件（即低温或高离子强度）会降低持续合成能力。大肠杆菌单链DNA结合蛋白可刺激基因5蛋白-硫氧还蛋白复合物的延伸速率和持续合成能力。
