Park K, Mukhopadhyay S, Chattoraj D K
Laboratory of Biochemistry, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
J Biol Chem. 1998 Sep 18;273(38):24906-11. doi: 10.1074/jbc.273.38.24906.
Origin opening is essential for the initiation of DNA replication in the theta mode and requires binding of initiator proteins. Using reactivity to KMnO4 in vivo as an assay, we find that, like initiation, origin opening of the Escherichia coli plasmid P1 requires the host initiators DnaA and HU and the plasmid-encoded initiator RepA. The ability to detect opening at the P1ori in vivo allowed us to study this activity at various copy numbers in chimeric replicons. The opening was prevented when the P1ori was cloned in high copy vectors or when excess RepA binding sites (iterons) were provided in trans. However, when RepA supply was also increased, the opening was efficient. A further increase in RepA prevented opening. Replication of an incoming P1 under these conditions correlated with opening. These results demonstrate that initiation is possible even at abnormally high origin concentrations and that oversupply of RepA, relative to iterons, can prevent replication by blocking origin opening. It appears that plasmid overreplication can be prevented either by limiting RepA or by accumulating RepA at a rate higher than that of the origin.
起始开放对于以θ模式启动DNA复制至关重要，并且需要起始蛋白的结合。利用体内对高锰酸钾的反应性作为一种检测方法，我们发现，与起始一样，大肠杆菌质粒P1的起始开放需要宿主起始蛋白DnaA和HU以及质粒编码的起始蛋白RepA。在体内检测P1ori处开放的能力使我们能够在嵌合复制子的各种拷贝数下研究这种活性。当P1ori克隆到高拷贝载体中或通过反式提供过量的RepA结合位点（迭代子）时，开放被阻止。然而，当RepA的供应也增加时，开放是有效的。RepA的进一步增加会阻止开放。在这些条件下，进入的P1的复制与开放相关。这些结果表明，即使在异常高的起始浓度下起始也是可能的，并且相对于迭代子而言，RepA的供应过剩可通过阻断起始开放来阻止复制。看来，要么通过限制RepA，要么通过以高于起始的速率积累RepA，都可以防止质粒过度复制。
