Yoo S K, Ito J
Department of Microbiology and Immunology, University of Arizona Health Sciences Center, Tucson 85724.
Virology. 1989 Jun;170(2):442-9. doi: 10.1016/0042-6822(89)90435-2.
A cell-free system has been developed from cells of an Escherichia coli strain, carrying cloned genes 1 and 8 of bacteriophage PRD1, that catalyzes protein-primed DNA synthesis. DNA synthesis in vitro is entirely dependent upon the addition of PRD1 DNA-protein complex as template, Mg2+, and four deoxyribonucleoside triphosphates. No in vitro DNA synthesis was observed when deproteinized PRD1 DNA was used as template. The origin and direction of PRD1 DNA replication in vitro was determined by restriction enzyme analysis of 32P-labeled PRD1 DNA synthesized in this system. Replication starts at both ends of the linear PRD1 DNA template. Alkaline sucrose gradient centrifugation and agarose gel electrophoresis showed that full-length PRD1 DNA is synthesized in vitro. DNA synthesis in this system is inhibited by the drug aphidicolin. We also observed that dimethyl sulfoxide (DMSO) stimulates in vitro DNA synthesis, although it inhibits bacterial DNA polymerase.
已从携带噬菌体PRD1克隆基因1和8的大肠杆菌菌株细胞中开发出一种无细胞系统，该系统可催化蛋白质引发的DNA合成。体外DNA合成完全依赖于添加PRD1 DNA-蛋白质复合物作为模板、Mg2+和四种脱氧核糖核苷三磷酸。当使用脱蛋白的PRD1 DNA作为模板时，未观察到体外DNA合成。通过对该系统中合成的32P标记的PRD1 DNA进行限制性酶切分析，确定了体外PRD1 DNA复制的起始点和方向。复制从线性PRD1 DNA模板的两端开始。碱性蔗糖梯度离心和琼脂糖凝胶电泳表明，体外合成了全长PRD1 DNA。该系统中的DNA合成受到药物阿非科林的抑制。我们还观察到，二甲基亚砜（DMSO）刺激体外DNA合成，尽管它抑制细菌DNA聚合酶。
