Antiparallel plasmid-plasmid pairing may control P1 plasmid replication.
Antiparallel plasmid-plasmid pairing may control P1 plasmid replication.
复制标题
反平行质粒-质粒配对可以控制 P1 质粒复制。
DOI：
10.1073/pnas.88.20.9011
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发表时间：
1991
影响因子：
11.1
通讯作者：
Austin,SJ
中科院分区：
文献类型：
--
作者：
Abeles,AL;Austin,SJ
P1质粒复制子的拷贝数受到严格控制，每个新生细胞只能复制一到两个拷贝。控制是通过复制控制位点incA的作用来实现的，incA包含9个重复的19个碱基对结合位点，用于质粒编码的启动蛋白RepA。一组5个相似的重复出现在RepA触发起始的复制原点。使用由大肠杆菌提取物、P1原点作为模板和纯化的RepA蛋白组成的体外复制系统，我们发现含有incA位点块原点的超卷曲DNA环在反式中具有功能。关闭以原点与incA序列的1:1比例完成。这不是由于RepA蛋白的滴定，因为过量的RepA可以添加而不恢复活性。相反，incA序列似乎通过质粒-质粒配对事件中的直接接触来阻断起源。当起源位点和incA位点同时存在于一个质粒上时，与子分子的反式接触似乎比顺式环更占优势。结果与复制控制模型一致，其中子质粒通过配对来阻止自己的复制，其中每个起源都与其伴侣的incA位点接触。
The copy number of the P1 plasmid replicon is stringently controlled, giving only one or two copies per newborn cell. Control is achieved by the action of the copy-control locus incA, which contains nine repeats of the 19-basepair binding site for the plasmid-encoded initiator protein RepA. A set of five similar repeats are present in the replication origin where RepA acts to trigger initiation. Using an in vitro replication system consisting of an Escherichia coli extract, the P1 origin as a template, and purified RepA protein, we show that supercoiled DNA circles containing the incA locus block origin function in trans. Shutdown becomes complete at a 1:1 ratio of origin to incA sequences. This is not due to titration of the RepA protein, as an excess of RepA can be added without restoring activity. Rather, the incA sequences appear to block the origin by direct contact in a plasmid-plasmid pairing event. When both the origin and the incA locus are present on one plasmid, trans contacts with daughter molecules appear to predominate over cis looping. The results are consistent with a model for replication control where daughter plasmids block their own replication by a pairing in which each origin is in contact with the incA locus of its partner.