The functional analysis of YabA, which interacts with DnaA and regulates initiation of chromosome replication in Bacillus subtils
The functional analysis of YabA, which interacts with DnaA and regulates initiation of chromosome replication in Bacillus subtils
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DOI：
10.1266/ggs.83.111
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发表时间：
2008-04-01
影响因子：
1.1
通讯作者：
Ishikawa, Shu
中科院分区：
文献类型：
--
作者：
Cho, Eunha;Ogasawara, Naotake;Ishikawa, Shu
细菌染色体DNA复制的启动及其调控是关键事件。DNAA是启动DNA复制所必需的，在细菌中是保守的。在大肠杆菌中，HDA通过与DNAA和DNAN形成三元复合体，促进ATP-DNAA的水解，确保DNAA在复制周期中及时失活。在枯草芽孢杆菌中，YABA还与DNAA和DNAN形成三元复合体，负调控DNA复制的起始步骤。然而，Yaba与HDA在结构上没有同源性，其调控机制本身也没有得到澄清。在这里，与HDA相反，我们观察到DNAA转录在Yaba的低表达和过表达期间是稳定的。CHA-P-CHIP分析表明，YABA的缺失不影响DNAA与DNA的结合。另一方面，酵母双杂交分析表明，DNAA-ATP结合区与Yaba相互作用。此外，通过酵母双杂交分析分离的Yaba相互作用缺陷突变体的突变位于ATP结合结构域的后面，而HDA被认为与ATP结合口袋本身相互作用。在枯草芽孢杆菌中引入Dna A(Y144C)突变，使其与Yaba的相互作用失效，但不影响与Dna A本身或与Dna D的相互作用，导致复制的过度启动和异步启动，并阻止了Yaba焦点的形成，进一步证明了位于ATP结合口袋对面的氨基酸对Yaba结合是重要的。这些结果表明，Yaba确实通过一种不同于HDA在E.ColiRIDA系统中使用的机制来调控DNA复制的启动。有趣的是，所有YABA结合缺陷的DNAA突变体在酵母双杂交实验中也显示DNAD结合减少，这表明YABA可以通过竞争性地抑制DNAD与DNAA的结合来抑制复制启动。
The initiation of bacterial chromosome DNA replication and its regulation are critical events. DnaA is essential for initiation of DNA replication and is conserved throughout bacteria. In Escherichia coli, hydrolysis of ATP-DnaA is promoted by Hda through formation of a ternary complex with DnaA and DnaN, ensuring the timely inactivation of DnaA during the replication cycle. In Bacillus subtilis, YabA also forms a ternary complex with DnaA and DnaN, and negatively regulates the initiation step of DNA replication. However, YabA shares no structural homology with Hda and the regulatory mechanism itself has not been clarified. Here, in contrast to Hda, we observed that dnaA transcription was stable during under- and overexpression of YabA. ChA-P-chip assays showed that the depletion of YabA did not affect DNA binding by DnaA. On the other hand, yeast two-hybrid analysis indicated that the DnaA ATP-binding domain interacts with YabA. Moreover, mutations in YabA interaction-deficient mutants, isolated by yeast two-hybrid analysis, are located at the back of the ATP-binding domain, whereas Hda is thought to interact with the ATP-binding pocket itself. The introduction into B. subtilis of a dnaA(Y144C) mutation, which disabled the interaction with YabA but did not affect interactions either with DnaA itself or with DnaD, resulted in over-initiation and asynchronous initiation of replication and disabled the formation of YabA foci, further demonstrating that the amino acid on the opposite side to the ATP-binding pocket is important for YabA binding. These results indicate that YabA indeed regulates the initiation of DNA replication by a different mechanism from that used by Hda in the E. coli RIDA system. Interestingly, all DnaA mutants deficient in YabA binding also displayed reduced DnaD binding in yeast two-hybrid assays, suggesting that YabA can inhibit replication initiation through competitive inhibition of DnaD binding to DnaA.