Khan S A, Murray R W, Koepsel R R
Department of Microbiology, Biochemistry and Molecular Biology, University of Pittsburgh, School of Medicine, PA 15261.
Biochim Biophys Acta. 1988 Dec 20;951(2-3):375-81. doi: 10.1016/0167-4781(88)90109-1.
The origin of replication of plasmid pT181 is nicked by the plasmid-encoded RepC protein. This nick presumably serves as the start-site of pT181 replication by extension 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. In vitro replication of a recombinant plasmid carrying two pT181 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 PT181 origins in inverted orientation resulted in initiation of replication at one origin and stalling of the replisome at the other origin. 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。这个切口大概通过延伸合成作为pT181复制的起始位点。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前导链相对应。携带两个直接定向的pT181复制起点的重组质粒的体外复制显示，产生了环状单链DNA，其对应于在一个起点序列处起始复制并在另一个起点处终止复制。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，两个反向的PT181复制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。
