耐辐射奇球菌中PprI蛋白的单分子动态作用机制

Single-molecule dynamic mechanism of PprI in Deinococcus radiodurans

摘要:

【背景】 在日益严重的环境污染中，越来越多的生物受到核辐射、化学污染、生物污染等危害，严重影响到生态系统的平衡，而耐辐射奇球菌(Deinococcus radiodurans)具有强大的DNA修复能力，使它能在各种极端环境下生存。PprI蛋白作为D. radiodurans中DNA损伤修复过程的开关，异源表达后可以显著提高其他真核和原核生物在极端环境下的生存率。目前对PprI蛋白的研究大多局限于传统生化手段，在活细胞内实时动态观测单个PprI蛋白的反应过程仍然相对滞后。【目的】 探究DNA损伤前后PprI蛋白在单分子水平的动态变化，从单分子角度精准揭示PprI蛋白在DNA损伤修复中的关键作用，为研究耐辐射奇球菌DNA修复机制奠定理论基础。【方法】 利用光转换荧光蛋白mMaple3原位标记的PprI蛋白，通过基于全内反射荧光(total internal reflection fluorescent, TIRF)显微成像的单分子示踪光激活定位显微镜(single-particle tracking photoactivated localization microscopy, sptPALM)技术，持续激活低密度的mMaple3荧光蛋白，实现活细胞内PprI蛋白的单分子定位与示踪，明确PprI蛋白在DNA损伤前后的分子动力学特征。【结果】 通过对PprI蛋白表观扩散系数分布的拟合，将其分为3种运动状态，即固定态(D^*=0.07 μm²/s)、缓慢扩散态(D^*=0.21 μm²/s)和快速扩散态(D^*=0.65μm²/s)。发现在DNA受到损伤后的细胞中，PprI蛋白扩散态分子比例显著上升，而其固定态分子比例显著下降。【结论】 利用单分子示踪技术精确表征了PprI蛋白在DNA受损后大部分蛋白运动速率偏向快速运动，表面DNA损伤释放了较大比例的结合态PprI蛋白。本研究可以深化PprI介导的DNA损伤修复系统的分子机制模型，也可以为利用单分子技术研究其他DNA修复反应提供技术参考。

Abstract:

[Background] Amid the aggravating environmental pollution, increasing organisms are endangered by nuclear radiation, chemical pollution, and biological pollution, which seriously disrupts the balance of ecosystems. Deinococcus radiodurans with remarkable performance of DNA repair can survive in various extreme environments. PprI acts as a switch in the DNA repair process in D.radiodurans, and the heterologous expression of this protein can significantly enhance the survival rates of other eukaryotes and prokaryotes in extreme conditions. The available studies on PprI molecules are predominantly conducted with biochemical methods, and the real-time dynamic observation on reactions of single PprI molecules in living cells remains underdeveloped. [Objective] We explored the dynamics of PprI at the single-molecule level before and after DNA damage and revealed the role of PprI in DNA repair, aiming to enhance our understanding of the DNA repair mechanism in D.radiodurans. [Methods] The PprI molecules of D.radiodurans were labeled with the photo-activated fluorescent protein mMaple3. The low-density mMaple3 fluorescent protein was continuously activated by single-particle tracking photoactivated localization microscopy (sptPALM) based on total internal reflection fluorescent (TIRF) microimaging to realize the single-molecule localization and tracking of PprI in living cells, on the basis of which the molecular dynamics of PprI molecules before and after DNA damage was clarified. [Results] By analyzing the distribution of the apparent diffusion coefficients of PprI molecules, we identified three distinct species: immobile molecules (D^*=0.07 μm²/s), slow-diffusing molecules (D^*=0.21 μm²/s), and fast-diffusing molecules (D^*=0.65 μm²/s). After DNA damage, we observed a significant increase in the proportion of diffusing PprI molecules and a significant decrease in the proportion of immobile molecules. [Conclusion] Using the single-molecule tracking technique, we accurately characterized the movement of PprI molecules, finding that most PprI molecules moved fast after DNA damage. Additionally, DNA damage at the surface released a large proportion of immobile PprI molecules. This study improves the molecular mechanism model of the PprI-mediated DNA repair system and paves a way for studying other DNA repair reactions using single-molecule techniques.