Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Department of Microbiology, Weigla 12, 53-114 Wrocław, Poland.
Max Planck Institute for Molecular Genetics, Ihnestrasse 63-73, 14195 Berlin, Germany.
J Mol Biol. 2014 Jul 29;426(15):2769-82. doi: 10.1016/j.jmb.2014.05.018. Epub 2014 May 24.
In bacteria, chromosome replication is initiated by binding of the DnaA initiator protein to DnaA boxes located in the origin of chromosomal replication (oriC). This leads to DNA helix opening within the DNA-unwinding element. Helicobacter pylori oriC, the first bipartite origin identified in Gram-negative bacteria, contains two subregions, oriC1 and oriC2, flanking the dnaA gene. The DNA-unwinding element region is localized in the oriC2 subregion downstream of dnaA. Surprisingly, oriC2-DnaA interactions were shown to depend on DNA topology, which is unusual in bacteria but is similar to initiator-origin interactions observed in higher organisms. In this work, we identified three DnaA boxes in the oriC2 subregion, two of which were bound only as supercoiled DNA. We found that all three DnaA boxes play important roles in orisome assembly and subsequent DNA unwinding, but different functions can be assigned to individual boxes. This suggests that the H. pylori oriC may be functionally divided, similar to what was described recently for Escherichia coli oriC. On the basis of these results, we propose a model of initiation complex formation in H. pylori.
在细菌中，染色体复制是通过 DnaA 起始蛋白与位于染色体复制起点（oriC）的 DnaA 盒结合而启动的。这导致 DNA 解旋元件内的 DNA 螺旋打开。幽门螺杆菌 oriC 是革兰氏阴性细菌中第一个双分区起点，包含两个亚区，oriC1 和 oriC2，位于 dnaA 基因的两侧。DNA 解旋元件区域位于 dnaA 下游的 oriC2 亚区。令人惊讶的是，oriC2-DnaA 相互作用依赖于 DNA 拓扑结构，这在细菌中是不寻常的，但与在高等生物中观察到的起始子-原点相互作用相似。在这项工作中，我们在 oriC2 亚区中鉴定出三个 DnaA 盒，其中两个仅与超螺旋 DNA 结合。我们发现，所有三个 DnaA 盒在 ori 体组装和随后的 DNA 解旋中都发挥重要作用，但可以为各个盒分配不同的功能。这表明幽门螺杆菌 oriC 可能在功能上是分开的，类似于最近描述的大肠杆菌 oriC。基于这些结果，我们提出了幽门螺杆菌起始复合物形成的模型。
