Zhao A C, Khan S A
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pennsylvania 15261, USA.
J Bacteriol. 1996 Sep;178(17):5222-8. doi: 10.1128/jb.178.17.5222-5228.1996.
pT181 and related plasmids of gram-positive bacteria replicate by a rolling-circle mechanism. The replication initiator protein of pT181, RepC, has origin-specific nicking-closing activities. Replication of the plasmid pT181 leading strand initiates by covalent extension of the RepC-generated nick, and the origin of replication contains signals for both initiation and termination of DNA replication. We have investigated the sequence requirements for the initiation and termination steps by using plasmids containing two pT181 origins. In vitro replication experiments showed that 18- and 24-bp synthetic oligonucleotides containing the RepC nick site were active in the termination of replication. However, initiation of replication required a larger region which also includes the RepC binding site. Plasmids containing the 18- and 24-bp region were also found to be nicked by the RepC protein. Our results demonstrate that sequence requirements for initiation and termination of pT181 replication overlap, but while the RepC binding site is required for initiation, it is dispensable for termination.
革兰氏阳性菌的pT181及相关质粒通过滚环机制进行复制。pT181的复制起始蛋白RepC具有位点特异性的切口闭合活性。质粒pT181前导链的复制通过RepC产生的切口的共价延伸起始，并且复制起点包含DNA复制起始和终止的信号。我们通过使用含有两个pT181起点的质粒研究了起始和终止步骤的序列要求。体外复制实验表明，含有RepC切口位点的18个碱基对和24个碱基对的合成寡核苷酸在复制终止中具有活性。然而，复制起始需要一个更大的区域，该区域还包括RepC结合位点。还发现含有18个碱基对和24个碱基对区域的质粒被RepC蛋白切口。我们的结果表明，pT181复制起始和终止的序列要求重叠，但是虽然RepC结合位点是起始所必需的，但对于终止却是可有可无的。
