Cooperative DnaA Binding to the Negatively Supercoiled datA Locus Stimulates DnaA-ATP Hydrolysis
Cooperative DnaA Binding to the Negatively Supercoiled datA Locus Stimulates DnaA-ATP Hydrolysis
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DOI：
10.1074/jbc.m116.762815
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发表时间：
2017-01-27
影响因子：
4.8
通讯作者：
Katayama, Tsutomu
中科院分区：
文献类型：
--
作者：
Kasho, Kazutoshi;Tanaka, Hiroyuki;Katayama, Tsutomu
大肠杆菌中复制的及时启动需要复制起始子ATP-DnaA的功能调节。ATP-DnaA的细胞水平在起始前增加，之后其水平通过DnaA结合的ATP的水解而降低，产生起始失活的ADP-DnaA。以前，我们报道了一种新的DnaA-ATP水解系统涉及染色体位点datA，并命名为datA依赖的DnaA-ATP水解（DDAH）。datA基因座包含一个核苷相关因子整合宿主因子（IHF）的结合位点和一组三个已知的DnaA结合位点，这些位点对DDAH很重要。然而，datA-IHF.DnaA复合物的形成和调控机制尚不清楚。我们现在证明，一个新的DnaA框datA内是必不可少的ATP-DnaA复合物的形成和DnaA-ATP水解。特定的DnaA残基，这是重要的相互作用与结合ATP和头-尾间的DnaA相互作用，也需要ATP-DnaA特异性寡聚体形成datA。此外，我们表明，负DNA超螺旋的datA稳定ATP-DnaA寡聚体，并刺激datA-IHF相互作用和DnaA-ATP水解。通过加入新生霉素（一种DNA促旋酶抑制剂）松弛DNA超螺旋，抑制细胞中的datA功能。在此基础上，我们提出了DDAH的datA-IHF、DnaA复合物形成和DNA超卷曲依赖性调控的机制模型。
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.