Initiator titration as a mechanism for regulation of initiation of DNA replication

大肠杆菌染色体的复制起始于一个独特的位点oriC。在一个细胞周期中，所有oriC位点都发生一次且仅发生一次复制。确保循环起始事件通过染色体位点datA操作的机制，datA是位于遗传图谱上94.7分钟处的1-kb片段，并且具有异常大量的细菌起始蛋白DnaA。缺乏datA的菌株正常生长，但由于额外的起始事件而表现出异步起始表型。检测了7个其它的DNA结合位点，以确定它们可能参与复制起始的控制。中断的七个网站并不影响复制的启动时间，即使当所有的同时中断。因此，datA似乎是一个独特的染色体元件，似乎在细菌细胞周期中的固定时间调节单个起始事件的游离和结合DnaA之间的平衡。在oriC螯合过程中，由oriC中的半甲基化GATC序列和SeqA蛋白介导的DnaA向新复制的datA的滴定将有助于防止oriC被去螯合时的重新启动。datA中第二或第三个DnaA盒（9 bp DnaA结合序列）的突变足以诱导突变表型。datA中的其他三个DnaA框似乎对datA功能没有影响。第二和第三个DnaA盒可以作为DnaA与整个datA区域合作结合的核心。DnaA的调节失活（RIDA）是另一种已知的防止不合时宜的额外启动的机制。我们发现RIDA的作用不依赖于DnaA对datA的滴定，这表明这两种机制可能在细胞周期中发挥互补作用，以确保预定的启动。

Replication of the Escherichia coli chromosome is initiated at a unique site, oriC.Concturrent initiations occur at all oriC sites present in a cell once and only once per cell cycle. A mechanism to ensure the cyclic initiation events operates through the chromosomal site, datA, which is a 1-kb segment located at 94.7 min on the genetic map and titrates exceptionally large amounts of the bacterial initiator protein, DnaA.A strain lacking datA grew normally but exhibited the asynchronous initiation phenotype due to extra initiation events.In the present study, seven other DnaA-binding sites were examined for their possible involvement in the control of replication initiation. Disruption of the seven sites did not affect the timing of initiation of replication, even when all of them were disrupted simultaneously. Thus, datA seems to be a unique chromosomal element that appears to adjust a balance between free and bound DnaA for the single initiation event at a fixed time in the bacterial cell cycle. Titration of DnaA to newly duplicated datA during oriC sequestration, which is mediated by hemimethylated GATC sequences in oriC and the SeqA protein, would contribute to prevent reinitiations when oriC is desequestered.Mutation either in the second or in the third DnaA box (a 9-bp DnaA-binding sequence) in datA were enough to induce the mutant phenotype. Other three DnaA boxes in datA seemed to have no effect on the datA function. The second and the third DnaA boxes may act as cores for the cooperative binding of DnaA to the entire datA region.Regulatory inactivation of DnaA (RIDA) is another mechanism known to prevent untimely extra initiations. We found that RIDA operates independent from DnaA titration to datA.This suggests that these two mechanisms may play complementary roles during the cell cycle to ensure the scheduled initiation.