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

引发剂滴定作为 DNA 复制起始调节机制

基本信息

批准号：
11680675
负责人：
OGAWA Tohru
金额：
$ 1.86万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680675/
关键词：
DNA replication DnaA oriC initiation of replication datA 複製イニシエーター大腸菌染色体

项目摘要

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.

大肠杆菌染色体的复制在一个独特的位点 oriC 处启动。细胞中存在的所有 oriC 位点同时启动，每个细胞周期仅发生一次。确保循环起始事件通过染色体位点 datA 运行的机制，datA 是位于遗传图谱上 94.7 分钟处的 1-kb 片段，并滴定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。在本研究中，检查了其他七个 DnaA 结合位点是否可能参与复制起始的控制。七个位点的破坏并不影响复制开始的时间，即使它们同时被破坏。因此，datA 似乎是一种独特的染色体元件，它似乎可以调节细菌细胞周期中固定时间的单个起始事件的游离和结合 DnaA 之间的平衡。在 oriC 隔离过程中，通过 oriC 中的半甲基化 GATC 序列和 SeqA 蛋白介导 DnaA 滴定到新复制的 datA，将有助于防止 oriC 解隔离时重新启动。第二个或第三个 DnaA 盒中的突变（9- datA 中的 bp DnaA 结合序列）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。第二个和第三个 DnaA 盒可以作为 DnaA 与整个 datA 区域协同结合的核心。DnaA 的调节失活 (RIDA) 是已知的另一种防止不合时宜的额外启动的机制。我们发现RIDA的运作独立于DnaA滴定到datA。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。