质粒R6K体外复制对一种质粒编码蛋白的需求
Requirement of a plasmid-encoded protein for replication in vitro of plasmid R6K.
作者信息
Inuzuka M, Helinski D R
出版信息
Proc Natl Acad Sci U S A. 1978 Nov;75(11):5381-5. doi: 10.1073/pnas.75.11.5381.
Abstract
Conditions are described for the replication of exogeneous R6K DNA in an in vitro system prepared from Escherichia coli cells. Replication of plasmid DNA in this system is semiconservative and sensitive to actinomycin D, novobiocin, arabinofuranosyl-CTP,N-ethylmaleimide, and inhibitors of DNA-dependent RNA polymerase. An ammonium sulfate fraction prepared from cells carrying the R6K plasmid is required for replication. A direct role in replication for a plasmid-encoded protein, designated pi, in this fraction is indicated by the inactivity of this fraction when prepared from cells carrying a temperature-sensitive mutant plasmid and the thermolability of this fraction when prepared from cells carrying a partial revertant of the mutant plasmid. This plasmid-encoded protein is necessary for the initiation of R6K DNA replication and functions before or during the formation of nascent RNA in the initiation process. The results of titration assays of this protein using various template DNAs suggest that the protein interacts with the plasmid DNA at the region essential for DNA replication.
摘要
本文描述了在由大肠杆菌细胞制备的体外系统中，外源R6K DNA复制的条件。该系统中质粒DNA的复制是半保留的，且对放线菌素D、新生霉素、阿拉伯糖基-CTP、N-乙基马来酰亚胺以及DNA依赖性RNA聚合酶抑制剂敏感。携带R6K质粒的细胞制备的硫酸铵分级分离物是复制所必需的。从携带温度敏感突变体质粒的细胞制备该分级分离物时其无活性，以及从携带突变质粒部分回复体的细胞制备该分级分离物时其具有热不稳定性，表明该分级分离物中一种由质粒编码的名为π的蛋白质在复制中起直接作用。这种由质粒编码的蛋白质对于R6K DNA复制的起始是必需的，并且在起始过程中新生RNA形成之前或期间发挥作用。使用各种模板DNA对该蛋白质进行滴定分析的结果表明，该蛋白质在DNA复制所必需的区域与质粒DNA相互作用。
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