CHARACTERIZATION AND PURIFICATION OF A PHAGE PHI-29-ENCODED DNA-POLYMERASE REQUIRED FOR THE INITIATION OF REPLICATION
CHARACTERIZATION AND PURIFICATION OF A PHAGE PHI-29-ENCODED DNA-POLYMERASE REQUIRED FOR THE INITIATION OF REPLICATION
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DOI：
10.1073/pnas.81.17.5325
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发表时间：
1984-01-01
期刊：
影响因子：
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通讯作者：
SALAS, M
中科院分区：
文献类型：
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作者：
BLANCO, L;SALAS, M
从携带含有基因2的重组质粒的大肠杆菌细胞中纯化形成蛋白质p3-dAMP起始复合物所需的噬菌体v φ 29蛋白质p2。MW为68，000的纯化蛋白质p2具有特异性DNA聚合酶活性，当使用v φ 29 DNA-蛋白质p3作为模板时，该活性延长了p3-dAMP起始复合物。当与vphi 29突变体sus 2感染的枯草芽孢杆菌或含质粒的大肠杆菌互补时，纯化的蛋白质p2在催化起始反应中具有活性。提供蛋白质p3的大肠杆菌提取物，以v φ 29 DNA-蛋白质p3作为模板。当在互补测定中使用纯化的蛋白质p3时，形成非常少量的起始复合物;添加来自未感染的B的提取物。subtilis或E.大肠杆菌强烈地刺激了起始反应，表明除了蛋白质p2和p3以及v φ 29 DNA-蛋白质p3模板之外，p3-dAMP起始复合物的形成还需要一些宿主因子。结果表明，噬菌体v φ 29编码在蛋白质引发的DNA合成的起始步骤所需的DNA聚合酶。
The phage .vphi.29 protein p2, required for the formation of the protein p3-dAMP initiation complex, was purified from Escherichia coli cells harboring a gene 2-containing recombinant plasmid. The purified protein p2, of MW 68,000, had a specific DNA polymerase activity that elongated the p3-dAMP initiation complex when .vphi.29 DNA-protein p3 was used as template. The purified protein p2 was active in catalyzing the initiation reaction when complemented with .vphi.29 mutant sus2-infected Bacillis subtilis or plasmid-containing E. coli extracts providing protein p3, in the presence of .vphi.29 DNA-protein p3 as template. When purified protein p3 was used in the complementation assay, a very low amount of initiation complex was formed; addition of extracts from uninfected B. subtilis or E. coli strongly stimulated the initiation reaction, indicating that, in addition to proteins p2 and p3 and the .vphi.29 DNA-protein p3 template, some host factor(s) is required for the formation of the p3-dAMP initiation complex. The results show that phage .vphi.29 encodes a DNA polymerase that is required at the initiation step of protein-primed DNA synthesis.