Murray R W, Koepsel R R, Khan S A
Department of Microbiology, Biochemistry, and Molecular Biology, University of Pittsburgh School of Medicine, Pennsylvania 15261.
J Biol Chem. 1989 Jan 15;264(2):1051-7.
The origin of replication of plasmid pT181 is nicked by the plasmid-encoded RepC protein. The free 3'-hydroxyl end at the nick is presumably used as primer for leading strand DNA synthesis. In vitro replication of pT181 was found to generate single-stranded DNA in addition to the supercoiled, double-stranded DNA. The single-stranded DNA was circular and corresponded to the pT181 leading strand. Recombinant plasmids were constructed that contain two pT181 origins of replication in either direct or inverted orientation. In vitro replication of the plasmid carrying two origins in direct orientation was shown to generate circular, single-stranded DNA that corresponded to initiation of replication at one origin sequence and termination at the other origin. These results demonstrate that the origin of pT181 leading strand DNA replication also serves as the site for termination of replication. Interestingly, the presence of two origins in inverted orientation resulted in initiation of replication at one origin and stalling of the replisome at the other origin. These results suggest that RepC can reinitiate replication at the second origin by nicking partially replicated, relaxed DNA. These data are consistent with the replication of pT181 by a rolling circle mechanism and indicate that single-stranded DNA is an intermediate in pT181 replication.
质粒pT181的复制起点被质粒编码的RepC蛋白切开一个切口。切口处游离的3'-羟基末端大概被用作前导链DNA合成的引物。发现pT181的体外复制除了产生超螺旋双链DNA外，还产生单链DNA。单链DNA是环状的，与pT181的前导链相对应。构建了含有两个直接或反向排列的pT181复制起点的重组质粒。携带两个同向排列起点的质粒的体外复制显示产生了环状单链DNA，这与在一个起点序列处起始复制并在另一个起点处终止相对应。这些结果表明，pT181前导链DNA复制的起点也作为复制终止的位点。有趣的是，两个反向排列的起点的存在导致在一个起点处起始复制，而复制体在另一个起点处停滞。这些结果表明，RepC可以通过切开部分复制的松弛DNA在第二个起点重新起始复制。这些数据与pT181通过滚环机制进行复制一致，并表明单链DNA是pT181复制的中间体。
