Nozaki Shingo, Yamada Yoshitaka, Ogawa Tohru
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.
Genes Cells. 2009 Mar;14(3):329-41. doi: 10.1111/j.1365-2443.2008.01269.x. Epub 2009 Jan 12.
The initiation of replication in Escherichia coli is negatively controlled by a mechanism referred to as 'initiator titration', a process by which the initiator protein, DnaA, is titrated to newly replicated binding sequences on the chromosome to reduce the initiation potential for replication. Initiator titration occurs predominantly at the datA locus that binds exceptionally large amounts of DnaA molecules to prevent aberrant initiations. We found that this was enabled by integration host factor (IHF). Within datA, there is a consensus IHF recognition sequence between the two DnaA recognition sequences (DnaA boxes) essential for its function. Binding of IHF to this site was demonstrated both in vitro and in vivo. Disruption of the core sequence in the consensus of the IHF-binding resulted in increased origin concentration as observed in Delta datA cells. Furthermore, the number of DnaA molecules bound to datA was reduced in cells carrying a disruption in the IHF-binding core sequence. The IHF-binding site and the essential DnaA boxes had to be located at a proper distance and orientation to maintain the accurate initiation timing. Therefore, IHF is a unique element in the control of replication initiation that acts negatively at datA, while known to act as a positive regulator at oriC.
大肠杆菌中复制的起始受到一种称为“引发剂滴定”机制的负调控，在这个过程中，引发剂蛋白DnaA被滴定到染色体上新复制的结合序列上，以降低复制的起始潜力。引发剂滴定主要发生在datA位点，该位点能结合大量的DnaA分子以防止异常起始。我们发现这是由整合宿主因子（IHF）促成的。在datA内，其功能所必需的两个DnaA识别序列（DnaA框）之间存在一个共有IHF识别序列。体外和体内实验均证实了IHF与该位点的结合。IHF结合共有序列中的核心序列被破坏，会导致如在ΔdatA细胞中观察到的那样，起始点浓度增加。此外，携带IHF结合核心序列破坏的细胞中，与datA结合的DnaA分子数量减少。IHF结合位点和必需的DnaA框必须位于适当的距离和方向，以维持准确的起始时间。因此，IHF是复制起始控制中的一个独特元件，它在datA处起负调控作用，而在oriC处已知起正调控作用。
