SYNMIKRO, LOEWE Center for Synthetic Microbiology, Marburg, Germany.
Department of Chemistry, Philipps Universität Marburg, Marburg, Germany.
Sci Rep. 2018 Nov 6;8(1):16450. doi: 10.1038/s41598-018-34572-8.
Single-particle (molecule) tracking (SPT/SMT) is a powerful method to study dynamic processes in living bacterial cells at high spatial and temporal resolution. We have performed single-molecule imaging of early DNA double-strand break (DSB) repair events during homologous recombination in the model bacterium Bacillus subtilis. Our findings reveal that DNA repair centres arise at all sites on the chromosome and that RecN, RecO and RecJ perform fast, enzyme-like functions during detection and procession of DNA double strand breaks, respectively. Interestingly, RecN changes its diffusion behavior upon induction of DNA damage, from a largely diffusive to a DNA-scanning mode, which increases efficiency of finding all sites of DNA breaks within a frame of few seconds. RecJ continues being bound to replication forks, but also assembles at many sites on the nucleoid upon DNA damage induction. RecO shows a similar change in its mobility as RecN, and also remains bound to sites of damage for few hundred milliseconds. Like RecN, it enters the nucleoid in damaged cells. Our data show that presynaptic preparation of DSBs including loading of RecA onto ssDNA is highly rapid and dynamic, and occurs throughout the chromosome, and not only at replication forks or only at distinct sites where many breaks are processes in analogy to eukaryotic DNA repair centres.
单分子（分子）跟踪（SPT/SMT）是一种强大的方法，可在高时空分辨率下研究活细菌细胞中的动态过程。我们已经在模式细菌枯草芽孢杆菌中进行了同源重组过程中单链断裂（DSB）修复事件的单分子成像。我们的发现表明，DNA 修复中心出现在染色体的所有位置上，RecN、RecO 和 RecJ 在检测和加工 DNA 双链断裂时分别发挥快速、酶样的功能。有趣的是，RecN 在诱导 DNA 损伤后改变其扩散行为，从主要扩散转变为 DNA 扫描模式，这在几秒钟内增加了在一个框架内找到所有 DNA 断裂位点的效率。RecJ 继续结合复制叉，但在 DNA 损伤诱导时也会在核区的许多位点组装。RecO 的迁移率也像 RecN 一样发生变化，并且在几百毫秒内仍与损伤部位结合。与 RecN 一样，它进入受损细胞的核区。我们的数据表明，DSB 的预突触准备包括将 RecA 加载到 ssDNA 上，速度非常快且动态，并且发生在整个染色体上，而不仅仅是在复制叉上，或者仅在许多断裂过程中类似真核 DNA 修复中心的特定位置上。
