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

大肠杆菌染色体的复制是在一个独特的位点oriC开始的。在细胞中存在的所有ORIC位点上发生一次，并且每个细胞周期只发生一次。一种确保循环启动事件的机制通过染色体位置DATA进行操作，DATA是位于遗传图谱上94.7分钟的1kb片段，滴定异常大量的细菌启动蛋白DNAA。缺乏DNAA的菌株正常生长，但由于额外的启动事件而表现出异步启动表型。在本研究中，检测了其他7个DNAA结合位点，以确定它们可能参与复制启动的控制。七个位点的中断不影响复制开始的时间，即使所有这些站点都同时中断。因此，数据似乎是一种独特的染色体元件，似乎在细菌细胞周期的固定时间调节单个起始事件的自由和结合DNAA之间的平衡。在ORIC分离过程中，通过ORIC中半甲基化的GATC序列和SEQA蛋白将DNAA滴定到新复制的数据中，这将有助于防止ORIC脱序时的重新启动。数据中第二个或第三个DNAA盒(一个9个碱基的DNAA结合序列)中的突变足以诱导突变表型。数据中的其他三个DNAA框似乎对数据功能没有影响。第二和第三个DNAA盒可以作为DNAA与整个数据区协同结合的核心。DNAA(RIDA)的调节失活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独立于DNAA滴定对数据进行操作，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.