首页> 美国卫生研究院文献>The Journal of Biological Chemistry >Cooperative DnaA Binding to the Negatively Supercoiled datA Locus Stimulates DnaA-ATP Hydrolysis
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Cooperative DnaA Binding to the Negatively Supercoiled datA Locus Stimulates DnaA-ATP Hydrolysis

机译:协同DnaA绑定到负超螺旋datA基因座刺激DnaA-ATP水解。

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摘要

Timely initiation of replication in Escherichia coli requires functional regulation of the replication initiator, ATP-DnaA. The cellular level of ATP-DnaA increases just before initiation, after which its level decreases through hydrolysis of DnaA-bound ATP, yielding initiation-inactive ADP-DnaA. Previously, we reported a novel DnaA-ATP hydrolysis system involving the chromosomal locus datA and named it datA-dependent DnaA-ATP hydrolysis (DDAH). The datA locus contains a binding site for a nucleoid-associating factor integration host factor (IHF) and a cluster of three known DnaA-binding sites, which are important for DDAH. However, the mechanisms underlying the formation and regulation of the datA-IHF·DnaA complex remain unclear. We now demonstrate that a novel DnaA box within datA is essential for ATP-DnaA complex formation and DnaA-ATP hydrolysis. Specific DnaA residues, which are important for interaction with bound ATP and for head-to-tail inter-DnaA interaction, were also required for ATP-DnaA-specific oligomer formation on datA. Furthermore, we show that negative DNA supercoiling of datA stabilizes ATP-DnaA oligomers, and stimulates datA-IHF interaction and DnaA-ATP hydrolysis. Relaxation of DNA supercoiling by the addition of novobiocin, a DNA gyrase inhibitor, inhibits datA function in cells. On the basis of these results, we propose a mechanistic model of datA-IHF·DnaA complex formation and DNA supercoiling-dependent regulation for DDAH.
机译:大肠杆菌中复制的及时启动需要复制启动子ATP-DnaA的功能调节。 ATP-DnaA的细胞水平在启动之前就增加了,此后其水平通过与DnaA结合的ATP的水解而降低,从而产生了启动不活跃的ADP-DnaA。以前,我们报道了一种涉及染色体基因座datA的新型DnaA-ATP水解系统,并将其命名为datA依赖性DnaA-ATP水解(DDAH)。 datA基因座包含一个与核苷酸相关的因子整合宿主因子(IHF)的结合位点和三个已知的DnaA结合位点的簇,这对DDAH很重要。但是,尚不清楚datA-IHF·DnaA复合物形成和调控的机制。现在,我们证明datA中的新型DnaA框对于ATP-DnaA复合物的形成和DnaA-ATP的水解至关重要。特定的DnaA残基对于与结合的ATP相互作用以及头对尾的DnaA相互作用至关重要,也是datA上ATP-DnaA特异性寡聚物形成的必需残基。此外,我们显示,datA的负DNA超螺旋可稳定ATP-DnaA低聚物,并刺激datA-IHF相互作用和DnaA-ATP水解。通过添加新霉素(一种DNA回旋酶抑制剂)来放松DNA超螺旋,从而抑制细胞中的datA功能。根据这些结果,我们提出了datA-IHF·DnaA复合物形成的机制模型和DDAH的DNA超螺旋依赖性调控。

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