Wickner S H, Chattoraj D K
Proc Natl Acad Sci U S A. 1987 Jun;84(11):3668-72. doi: 10.1073/pnas.84.11.3668.
We have developed an in vitro DNA-replication system that replicates exogenously added mini-P1 plasmid DNA. The system consists of purified P1 RepA protein and a partially purified mixture of Escherichia coli replication proteins. It is essentially the same as that described for the replication of oriC plasmid DNA [Fuller, R.S., Kaguni, J.M. & Kornberg, A. (1981) Proc. Natl. Acad. Sci. USA 78, 7370-7374]. Mini-P1 DNA replication requires the E. coli DnaA initiation protein in addition to the P1 RepA initiation protein. The reaction is inhibited by rifampicin, novobiocin, and antibody to DnaB, suggesting the involvement of RNA polymerase, DNA gyrase, and DnaB protein. Replication is initiated in the region of the P1 origin of replication and proceeds unidirectionally as determined by electron microscopy. Thus, the in vitro system mimics the essential features of mini-P1 replication as suggested by genetic studies.
我们开发了一种体外DNA复制系统，该系统可复制外源添加的微型P1质粒DNA。该系统由纯化的P1 RepA蛋白和大肠杆菌复制蛋白的部分纯化混合物组成。它与用于oriC质粒DNA复制所描述的系统基本相同[富勒，R.S.，卡古尼，J.M.和科恩伯格，A.（1981年）美国国家科学院院刊78，7370 - 7374]。微型P1 DNA复制除了需要P1 RepA起始蛋白外，还需要大肠杆菌DnaA起始蛋白。该反应受到利福平、新生霉素和抗DnaB抗体的抑制，这表明RNA聚合酶、DNA回旋酶和DnaB蛋白参与其中。复制在P1复制起点区域起始，并如电子显微镜所确定的那样单向进行。因此，该体外系统模拟了遗传学研究中所提示的微型P1复制的基本特征。
