Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
Nucleic Acids Res. 2010 May;38(9):2851-64. doi: 10.1093/nar/gkp1236. Epub 2010 Jan 13.
Initiation of bacterial DNA replication at oriC is mediated by primosomal proteins that act cooperatively to melt an AT-rich region where the replicative helicase is loaded prior to the assembly of the replication fork. In Bacillus subtilis, the dnaD, dnaB and dnaI genes are essential for initiation of DNA replication. We established that their mRNAs are maintained in fast growing asynchronous cultures. DnaB is truncated at its C-terminus in a growth phase-dependent manner. Proteolysis is confined to cytosolic, not to membrane-associated DnaB, and affects oligomerization. Truncated DnaB is depleted at the oriC relative to the native protein. We propose that DNA-induced oligomerization is essential for its action at oriC and proteolysis regulates its localization at oriC. We show that DnaB has two separate ssDNA-binding sites one located within residues 1-300 and another between residues 365-428, and a dsDNA-binding site within residues 365-428. Tetramerization of DnaB is mediated within residues 1-300, and DNA-dependent oligomerization within residues 365-428. Finally, we show that association of DnaB with the oriC is asymmetric and extensive. It encompasses an area from the middle of dnaA to the end of yaaA that includes the AT-rich region melted during the initiation stage of DNA replication.
oriC 处细菌 DNA 复制的起始由原核起始蛋白介导，这些蛋白协同作用以融化富含 AT 的区域，复制解旋酶在复制叉组装之前加载到该区域。在枯草芽孢杆菌中，dnaD、dnaB 和 dnaI 基因对于 DNA 复制的起始是必不可少的。我们确定它们的 mRNA 在快速生长的非同步培养物中得以维持。DnaB 在生长阶段依赖性地在其 C 末端被截断。蛋白水解仅限于细胞质，而不是膜结合的 DnaB，并且影响寡聚化。与天然蛋白相比，截断的 DnaB 在 oriC 处被耗尽。我们提出 DNA 诱导的寡聚化对于其在 oriC 处的作用是必需的，并且蛋白水解调节其在 oriC 处的定位。我们表明 DnaB 具有两个独立的单链 DNA 结合位点，一个位于残基 1-300 内，另一个位于残基 365-428 之间，并且 dsDNA 结合位点位于残基 365-428 内。DnaB 的四聚化由残基 1-300 介导，而 DNA 依赖性寡聚化由残基 365-428 介导。最后，我们表明 DnaB 与 oriC 的结合是不对称且广泛的。它涵盖了从 dnaA 的中间到 yaaA 的末端的区域，包括在 DNA 复制起始阶段融化的富含 AT 的区域。
