Department of Molecular Microbiology, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland.
Department of Microbiology, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.
Microbiology (Reading). 2019 Dec;165(12):1365-1375. doi: 10.1099/mic.0.000859. Epub 2019 Oct 8.
DNA replication is controlled mostly at the initiation step. In bacteria, replication of the chromosome starts at a single origin of replication called . The initiator protein, DnaA, binds to specific sequences (DnaA boxes) within and assembles into a filament that promotes DNA double helix opening within the DNA unwinding element (DUE). This process has been thoroughly examined in model bacteria, including and but we have a relatively limited understanding of chromosomal replication initiation in other species. Here, we reveal new details of DNA replication initiation in , a group of Gram-positive soil bacteria that possesses a long linear (8-10 Mbps) and GC-rich chromosome with a centrally positioned . We used comprehensive and analyses to better characterize the structure of . We identified 14 DnaA-binding motifs and determined the consensus sequence of the DnaA box. Unexpectedly, our analysis using the WebSIDD algorithm revealed the presence of two putative DUEs (DUE1 and DUE2) located very near one another toward the 5' end of the region. P1 nuclease assay revealed that DNA unwinding occurs at both of the proposed sites, but using an replication initiation point mapping we were able to confirm only one of them (DUE2). The previously observed transcriptional activity of the region may help explain the current results. We speculate that transcription itself could modulate activity in by determining whether DNA unwinding occurs at DUE1 or DUE2.
DNA 复制主要在起始步骤受到控制。在细菌中，染色体的复制从称为 oriC 的单个复制起点开始。起始蛋白 DnaA 结合到oriC 内的特定序列（DnaA 盒）上，并组装成纤维，促进 DNA 解旋元件（DUE）内的 DNA 双螺旋打开。这个过程在模式细菌中已经得到了深入研究，包括大肠杆菌和枯草芽孢杆菌，但我们对其他物种的染色体复制起始的了解相对有限。在这里，我们揭示了革兰氏阳性土壤细菌 中的 DNA 复制起始的新细节，该细菌拥有一条长线性（8-10 Mbps）和 GC 丰富的染色体，其中心位置有一个 oriC。我们使用综合的 DNA 结合和 DNA 解旋分析来更好地描述 oriC 的结构。我们鉴定了 14 个 DnaA 结合基序，并确定了 DnaA 盒的共识序列。出乎意料的是，我们使用 WebSIDD 算法进行的 DNA 解旋分析揭示了两个假定的 DUE（DUE1 和 DUE2）非常靠近 oriC 区域的 5'端。P1 核酸酶分析显示，DNA 解旋发生在两个拟议的位点，但使用 复制起始点映射，我们只能确认其中一个（DUE2）。先前观察到的 区域的转录活性可能有助于解释当前的结果。我们推测，转录本身可以通过确定 DNA 解旋是否发生在 DUE1 或 DUE2 来调节 在 中的活性。
