Molecular Horizons Institute and School of Chemistry and Biomolecular Science, University of Wollongong, Wollongong, Australia.
Illawarra Health and Medical Research Institute, Wollongong, NSW 2500, Australia.
Nucleic Acids Res. 2019 Apr 8;47(6):2946-2965. doi: 10.1093/nar/gkz003.
In bacteria, genetic recombination is a major mechanism for DNA repair. The RecF, RecO and RecR proteins are proposed to initiate recombination by loading the RecA recombinase onto DNA. However, the biophysical mechanisms underlying this process remain poorly understood. Here, we used genetics and single-molecule fluorescence microscopy to investigate whether RecF and RecO function together, or separately, in live Escherichia coli cells. We identified conditions in which RecF and RecO functions are genetically separable. Single-molecule imaging revealed key differences in the spatiotemporal behaviours of RecF and RecO. RecF foci frequently colocalize with replisome markers. In response to DNA damage, colocalization increases and RecF dimerizes. The majority of RecF foci are dependent on RecR. Conversely, RecO foci occur infrequently, rarely colocalize with replisomes or RecF and are largely independent of RecR. In response to DNA damage, RecO foci appeared to spatially redistribute, occupying a region close to the cell membrane. These observations indicate that RecF and RecO have distinct functions in the DNA damage response. The observed localization of RecF to the replisome supports the notion that RecF helps to maintain active DNA replication in cells carrying DNA damage.
在细菌中，遗传重组是 DNA 修复的主要机制。RecF、RecO 和 RecR 蛋白被认为通过将 RecA 重组酶加载到 DNA 上而引发重组。然而，这一过程的生物物理机制仍知之甚少。在这里，我们使用遗传学和单分子荧光显微镜技术来研究 RecF 和 RecO 是否在活的大肠杆菌细胞中共同或分别发挥作用。我们确定了 RecF 和 RecO 功能在遗传上可分离的条件。单分子成像揭示了 RecF 和 RecO 时空行为的关键差异。RecF 焦点经常与复制体标记共定位。在 DNA 损伤的情况下，共定位增加并且 RecF 二聚化。大多数 RecF 焦点依赖于 RecR。相反，RecO 焦点很少出现，很少与复制体或 RecF 共定位，并且在很大程度上独立于 RecR。在 DNA 损伤的情况下，RecO 焦点似乎发生了空间重分布，占据了靠近细胞膜的区域。这些观察结果表明 RecF 和 RecO 在 DNA 损伤反应中具有不同的功能。RecF 定位于复制体的观察结果支持这样一种观点，即 RecF 有助于在携带 DNA 损伤的细胞中维持活跃的 DNA 复制。
