Division of Biosciences, College of Health and Life Sciences, Brunel University London, Uxbridge, UB8 3PH, UK.
Division of Biosciences, College of Health and Life Sciences, Brunel University London, Uxbridge, UB8 3PH, UK.
DNA Repair (Amst). 2018 Oct;70:37-48. doi: 10.1016/j.dnarep.2018.08.002. Epub 2018 Aug 19.
Bacterial chromosome duplication is initiated at a single origin (oriC). Two forks are assembled and proceed in opposite directions with high speed and processivity until they fuse and terminate in a specialised area opposite to oriC. Proceeding forks are often blocked by tightly-bound protein-DNA complexes, topological strain or various DNA lesions. In Escherichia coli the RecBCD protein complex is a key player in the processing of double-stranded DNA (dsDNA) ends. It has important roles in the repair of dsDNA breaks and the restart of forks stalled at sites of replication-transcription conflicts. In addition, ΔrecB cells show substantial amounts of DNA degradation in the termination area. In this study we show that head-on encounters of replication and transcription at a highly-transcribed rrn operon expose fork structures to degradation by nucleases such as SbcCD. SbcCD is also mostly responsible for the degradation in the termination area of ΔrecB cells. However, additional processes exacerbate degradation specifically in this location. Replication profiles from ΔrecB cells in which the chromosome is linearized at two different locations highlight that the location of replication termination can have some impact on the degradation observed. Our data improve our understanding of the role of RecBCD at sites of replication-transcription conflicts as well as the final stages of chromosome duplication. However, they also highlight that current models are insufficient and cannot explain all the molecular details in cells lacking RecBCD.
细菌染色体的复制是从单个起始点（oriC）开始的。两个叉子组装并以高速和高顺式进行相反方向的推进，直到它们融合并在与 oriC 相对的专门区域终止。前进的叉子经常被紧密结合的蛋白-DNA 复合物、拓扑应变或各种 DNA 损伤所阻断。在大肠杆菌中，RecBCD 蛋白复合物是处理双链 DNA（dsDNA）末端的关键因素。它在 dsDNA 断裂的修复和复制转录冲突位点处停滞的叉子的重新启动中发挥着重要作用。此外，ΔrecB 细胞在终止区域显示出大量的 DNA 降解。在这项研究中，我们表明，在高度转录的 rrn 操纵子处，复制和转录的正面相遇会使叉结构暴露于核酸酶（如 SbcCD）的降解之下。SbcCD 也主要负责 ΔrecB 细胞终止区域的降解。然而，其他过程会加剧该位置的降解。从线性化在两个不同位置的染色体的 ΔrecB 细胞中得到的复制图谱突出表明，复制终止的位置可能会对观察到的降解产生一些影响。我们的数据增进了对 RecBCD 在复制转录冲突位点以及染色体复制末期的作用的理解。然而，它们也强调了当前的模型是不充分的，无法解释缺乏 RecBCD 的细胞中的所有分子细节。
