Cho Eunha, Ogasawara Naotake, Ishikawa Shu
Graduate School of Information Science, Nara Institute of Science and Technology, Ikoma, Nara, Japan.
Genes Genet Syst. 2008 Apr;83(2):111-25. doi: 10.1266/ggs.83.111.
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. ChAP-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.
细菌染色体DNA复制的起始及其调控是关键事件。DnaA对于DNA复制的起始至关重要，并且在所有细菌中都保守存在。在大肠杆菌中，Hda通过与DnaA和DnaN形成三元复合物来促进ATP-DnaA的水解，从而确保在复制周期中DnaA及时失活。在枯草芽孢杆菌中，YabA也与DnaA和DnaN形成三元复合物，并对DNA复制的起始步骤起负调控作用。然而，YabA与Hda没有结构同源性，其调控机制本身尚未阐明。在此，与Hda不同，我们观察到在YabA过表达和低表达时，dnaA转录是稳定的。染色质免疫沉淀芯片（ChAP-chip）分析表明，YabA的缺失并不影响DnaA与DNA的结合。另一方面，酵母双杂交分析表明，DnaA的ATP结合结构域与YabA相互作用。此外，通过酵母双杂交分析分离得到的YabA相互作用缺陷型突变体中的突变位于ATP结合结构域的背面，而Hda被认为是与ATP结合口袋本身相互作用。将dnaA(Y144C)突变引入枯草芽孢杆菌中，该突变使DnaA与YabA的相互作用丧失，但不影响DnaA自身或与DnaD的相互作用，导致复制过度起始和异步起始，并使YabA焦点的形成受阻，进一步证明了ATP结合口袋对面的氨基酸对于YabA结合很重要。这些结果表明，YabA确实通过与大肠杆菌RIDA系统中Hda不同的机制来调控DNA复制的起始。有趣的是，所有缺乏与YabA结合能力的DnaA突变体在酵母双杂交试验中也显示出与DnaD结合能力的降低，这表明YabA可以通过竞争性抑制DnaD与DnaA的结合来抑制复制起始。
