Birch P, Khan S A
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, PA 15261.
Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):290-4. doi: 10.1073/pnas.89.1.290.
Plasmid pT181 is a 4437-base-pair, multicopy plasmid of Staphylococcus aureus that encodes tetracycline resistance. The replication of the leading strand of pT181 DNA initiates by covalent extension of a site-specific nick generated by the initiator protein at the origin of replication and proceeds by an asymmetric rolling circle mechanism. The origin of the leading strand synthesis also serves as the site for termination of replication. Replication of pT181 DNA in vivo and in vitro has been shown to generate a single-stranded intermediate that corresponds to the leading strand of the DNA. In vivo results have suggested that a palindromic sequence, palA, located near the leading strand termination site acts as the lagging strand origin. In this paper we report the development and characterization of an in vitro system for the replication of single-stranded pT181 DNA. Synthesis of the lagging strand of pT181 proceeded in the absence of the leading strand synthesis and did not require the pT181-encoded initiator protein, RepC. The replication of the lagging strand required RNA polymerase-dependent synthesis of an RNA primer. Replication of single-stranded pT181 DNA was found to be greatly stimulated in the presence of the palA sequence. We also show that palA acts as the lagging strand origin and that DNA synthesis initiates within this region.
质粒pT181是金黄色葡萄球菌的一种4437个碱基对的多拷贝质粒，编码四环素抗性。pT181 DNA前导链的复制通过起始蛋白在复制起点产生的位点特异性切口的共价延伸开始，并通过不对称滚环机制进行。前导链合成的起点也作为复制终止的位点。已证明pT181 DNA在体内和体外的复制会产生与DNA前导链相对应的单链中间体。体内结果表明，位于前导链终止位点附近的回文序列palA作为后随链起点。在本文中，我们报告了一种用于单链pT181 DNA复制的体外系统的开发和特性。pT181后随链的合成在没有前导链合成的情况下进行，并且不需要pT181编码的起始蛋白RepC。后随链的复制需要RNA聚合酶依赖性的RNA引物合成。发现在palA序列存在的情况下，单链pT181 DNA的复制受到极大刺激。我们还表明palA作为后随链起点，并且DNA合成在该区域内起始。
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