Lanka E, Scherzinger E, Günther E, Schuster H
Proc Natl Acad Sci U S A. 1979 Aug;76(8):3632-6. doi: 10.1073/pnas.76.8.3632.
An enzyme has been isolated from Escherichia coli strains harboring the I-like plasmid R64drd11, which is capable of initiating DNA synthesis on the circular, single-stranded DNA of phages phi X174, fd, and G4. In the conversion of these templates to duplex forms in vitro, the enzyme can substitute for the functions of E. coli dna B-dnaB-dnaC-dnaG proteins, E. coli RNA polymerase, and E. coli dnaG protein, respectively. The enzyme requires all four ribonucleoside triphosphates for optimal activity, although a combination of ATP, CTP, and GTP can almost completely satisfy the rNTP requirement. The enzyme appears to cooperate specifically with DNA polymerases III because single-stranded DNA-dependent synthesis takes place in extracts deficient in DNA polymerases I and II but not in extracts from a dnaZ mutant. Highly purified enzyme preparations consist mostly of two major polypeptides, Mr 140,000 and 180,000, when analyzed by sodium dodecyl sulfate gel electrophoresis. These polypeptides cosediment with the enzyme activity through a glycerol gradient with a sedimentation coefficient of 3.6 S. DNA priming activity in extracts of E. coli strains harboring the mutant plasmids R64drd11 or ColIdrd1, which are derepressed in functions of conjugational DNA transfer, severalfold higher than the activity from strains carrying the corresponding wild-type plasmid. This correlation suggests that the enzyme may play a role in conjugational DNA synthesis.
已从携带 I 类质粒 R64drd11 的大肠杆菌菌株中分离出一种酶，该酶能够在噬菌体 phi X174、fd 和 G4 的环状单链 DNA 上起始 DNA 合成。在体外将这些模板转化为双链形式的过程中，该酶可分别替代大肠杆菌 dna B-dnaB-dnaC-dnaG 蛋白、大肠杆菌 RNA 聚合酶和大肠杆菌 dnaG 蛋白的功能。该酶需要所有四种核糖核苷三磷酸以达到最佳活性，不过 ATP、CTP 和 GTP 的组合几乎可以完全满足对核糖核苷三磷酸的需求。该酶似乎与 DNA 聚合酶 III 特异性协同作用，因为在缺乏 DNA 聚合酶 I 和 II 的提取物中可发生单链 DNA 依赖性合成，而在 dnaZ 突变体的提取物中则不能。通过十二烷基硫酸钠凝胶电泳分析时，高度纯化的酶制剂主要由两种主要多肽组成，分子量分别为 140,000 和 180,000。这些多肽通过甘油梯度与酶活性一起沉降，沉降系数为 3.6 S。携带在接合性 DNA 转移功能上处于去阻遏状态突变质粒 R64drd11 或 ColIdrd1 的大肠杆菌菌株提取物中的 DNA 引发活性，比携带相应野生型质粒的菌株的活性高几倍。这种相关性表明该酶可能在接合性 DNA 合成中起作用。
