Hernández-Tamayo Rogelio, Graumann Peter L
SYNMIKRO, LOEWE Center for Synthetic Microbiology, Marburg, Germany.
Department of Chemistry, Philipps Universität Marburg, Hans-Meerwein-Straße 6, 35032, Marburg, Germany.
BMC Res Notes. 2019 Apr 11;12(1):219. doi: 10.1186/s13104-019-4252-x.
Little is known about the activity and dynamics of ATPase RarA in B. subtilis, proposed to act at stalled DNA replication forks due to DNA damage. We performed fluorescence microscopy time lapse experiments with a functional RarA-mVenus fusion to visualize the dynamics of RarA during conditions that generate DNA damage.
In exponentially growing cells, we observed that 15% of the cells contained single RarA-mV (mVenus fluorescent fusion) foci moving throughout the entire cell between 3 min intervals. This percentage remained constant at different time points, indicating that focus formation during unperturbed growth is maintained at about a constant rate. When cells were exposed to stress conditions, the population of cells containing RarA-mV foci tripled after 60 min. Cells exposed to two DNA-damaging drugs, to 5 mM MMS or to 0.5 mM HO, showed a similar type of response, with RarA-mVenus foci moving more slowly than during unperturbed growth. It is likely that RarA-mV contributes to the repair of HO-induced lesions, and to a minor extent to MMS-induced lesions. The presence of foci in growing cells suggests that RarA also plays a role during the cell cycle, at least in a fraction of cells, possibly contributing to heterogeneity of response to DNA damage.
关于枯草芽孢杆菌中ATP酶RarA的活性和动力学知之甚少，RarA被认为在因DNA损伤而停滞的DNA复制叉处发挥作用。我们用功能性RarA-mVenus融合蛋白进行了荧光显微镜延时实验，以观察在产生DNA损伤的条件下RarA的动力学。
在指数生长的细胞中，我们观察到15%的细胞含有单个RarA-mV（mVenus荧光融合蛋白）焦点，在3分钟的间隔内移动穿过整个细胞。这个百分比在不同时间点保持恒定，表明在未受干扰的生长过程中焦点形成以大约恒定的速率维持。当细胞暴露于应激条件下时，含有RarA-mV焦点的细胞群体在60分钟后增加了两倍。暴露于两种DNA损伤药物（5 mM甲磺酸甲酯或0.5 mM过氧化氢）的细胞表现出类似的反应类型，RarA-mVenus焦点的移动比未受干扰的生长过程中更慢。RarA-mV可能有助于修复过氧化氢诱导的损伤，并在较小程度上有助于修复甲磺酸甲酯诱导的损伤。生长细胞中焦点的存在表明RarA在细胞周期中也发挥作用，至少在一部分细胞中，可能有助于对DNA损伤反应的异质性。
