Nakai H, Richardson C C
J Biol Chem. 1986 Nov 15;261(32):15208-16.
Three proteins catalyze RNA-primed DNA synthesis on the lagging strand side of the replication fork of bacteriophage T7. Oligoribonucleotides are synthesized by T7 gene 4 protein, which also provides helicase activity. DNA synthesis is catalyzed by gene 5 protein of the phage, and processivity of DNA synthesis is conferred by Escherichia coli thioredoxin, a protein that is tightly associated with gene 5 protein. T7 DNA polymerase and gene 4 protein associate to form a complex that can be isolated by filtration through a molecular sieve. The complex is stable in 50 mM NaCl but is dissociated by 100 mM NaCl, a salt concentration that does not inhibit RNA-primed DNA synthesis. T7 DNA polymerase forms a stable complex with single-stranded M13 DNA at 50 mM NaCl as measured by gel filtration, and this complex requires 200 mM NaCl for dissociation, a salt concentration that inhibits RNA-primed DNA synthesis. Gene 4 protein alone does not bind to single-stranded DNA. In the presence of MgCl2 and dTTP or beta, gamma-methylene dTTP, a gene 4 protein-M13 DNA complex that is stable at 200 mM NaCl is formed. The affinity of DNA polymerase for both gene 4 protein and single-stranded DNA leads to the formation of a gene 4 protein-DNA polymerase-M13 DNA complex even in the absence of nucleoside triphosphates. However, the binding of each protein to DNA plays an important role in mediating the interaction of the proteins with each other. High concentrations of single-stranded DNA inhibit RNA-primed DNA synthesis by diluting the amount of proteins bound to each template and reducing the frequency of protein-protein interactions. Preincubation of gene 4 protein, DNA polymerase, and M13 DNA in the presence of dTTP forms protein-DNA complexes that most efficiently catalyze RNA-primed DNA synthesis in the presence of excess single-stranded competitor DNA.
三种蛋白质催化噬菌体T7复制叉后随链一侧的RNA引发的DNA合成。寡核糖核苷酸由T7基因4蛋白合成，该蛋白还具有解旋酶活性。DNA合成由噬菌体的基因5蛋白催化，DNA合成的持续性由大肠杆菌硫氧还蛋白赋予，硫氧还蛋白是一种与基因5蛋白紧密结合的蛋白质。T7 DNA聚合酶和基因4蛋白结合形成复合物，该复合物可通过分子筛过滤分离。该复合物在50 mM NaCl中稳定，但在100 mM NaCl中解离，100 mM NaCl的盐浓度并不抑制RNA引发的DNA合成。通过凝胶过滤测量，T7 DNA聚合酶在50 mM NaCl下与单链M13 DNA形成稳定复合物，该复合物需要200 mM NaCl才能解离，200 mM NaCl的盐浓度会抑制RNA引发的DNA合成。单独的基因4蛋白不与单链DNA结合。在MgCl2和dTTP或β,γ-亚甲基dTTP存在的情况下，会形成在200 mM NaCl下稳定的基因4蛋白-M13 DNA复合物。即使在没有核苷三磷酸的情况下，DNA聚合酶对基因4蛋白和单链DNA的亲和力也会导致形成基因4蛋白-DNA聚合酶-M13 DNA复合物。然而，每种蛋白质与DNA的结合在介导蛋白质之间的相互作用中起着重要作用。高浓度的单链DNA通过稀释与每个模板结合的蛋白质量并降低蛋白质-蛋白质相互作用的频率来抑制RNA引发的DNA合成。在dTTP存在的情况下，对基因4蛋白、DNA聚合酶和M13 DNA进行预孵育，会形成蛋白质-DNA复合物，在存在过量单链竞争DNA的情况下，该复合物能最有效地催化RNA引发的DNA合成。
