Kaguni L S, Kaguni J M, Ray D S
J Bacteriol. 1981 Feb;145(2):974-9. doi: 10.1128/jb.145.2.974-979.1981.
The involvement of the Escherichia coli rep protein in the replication of M13 chimeric deoxyribonucleic acids (DNAs) carrying the E. coli chromosomal DNA replication origin (oriC) has been examined. Previous studies indicate that the cloning of a 3,550-base-pair sequence of chromosomal DNA containing oriC into an M13 vector allows extensive replication of the M13 oriC chimeric DNA in an E. coli rep-3 mutant. We have extended these studies by preparing a 330-base-pair deletion that specifically deletes the oriC sequence in the M13 oriC DNAs, to demonstrate that the replication observed in the rep-3 host is dependent on the cloned origin. Thus, a DNA-unwinding enzyme other than the rep protein may be involved in the strand separation process accompanying replication which initiates at oriC in the M13 oriC chimeric DNAs and in the E. coli chromosome. The rep assay used for assessing the functionality of the cloned oriC is useful for analysis of any rep-independent origin of replication functional in E. coli. A direct selection for a cloned origin of replication is possible in the rep-3 recA56 host. Since the cloned origin is nonessential for propagation of the M13 chimeric phage in a rep+ host, mutations in the cloned origin may be constructed, and the mutant phage may be examined by a simple transductional analysis of the rep-3 recA56 mutant strain.
已对大肠杆菌rep蛋白在携带大肠杆菌染色体DNA复制起点（oriC）的M13嵌合脱氧核糖核酸（DNA）复制中的作用进行了研究。先前的研究表明，将包含oriC的3550个碱基对的染色体DNA序列克隆到M13载体中，可使M13 oriC嵌合DNA在大肠杆菌rep - 3突变体中大量复制。我们通过制备一个330个碱基对的缺失片段来扩展这些研究，该缺失片段专门删除M13 oriC DNA中的oriC序列，以证明在rep - 3宿主中观察到的复制依赖于克隆的起点。因此，除rep蛋白外的一种DNA解旋酶可能参与了伴随复制的链分离过程，该复制在M13 oriC嵌合DNA和大肠杆菌染色体中的oriC处起始。用于评估克隆的oriC功能的rep检测法，对于分析在大肠杆菌中起作用的任何不依赖rep的复制起点很有用。在rep - 3 recA56宿主中可以直接选择克隆的复制起点。由于克隆的起点对于M13嵌合噬菌体在rep +宿主中的繁殖不是必需的，因此可以构建克隆起点中的突变，并且可以通过对rep - 3 recA56突变菌株进行简单的转导分析来检查突变噬菌体。
