Sisario Dmitri, Memmel Simon, Doose Sören, Neubauer Julia, Zimmermann Heiko, Flentje Michael, Djuzenova Cholpon S, Sauer Markus, Sukhorukov Vladimir L
Department of Biotechnology and Biophysics, University of Würzburg, Würzburg, Germany.
Department of Radiation Oncology, University Hospital of Würzburg, Würzburg, Germany.
FASEB J. 2018 Jun 12:fj201701435. doi: 10.1096/fj.201701435.
Induction of DNA double-strand breaks (DSBs) by ionizing radiation leads to formation of micrometer-sized DNA-repair foci, whose organization on the nanometer-scale remains unknown because of the diffraction limit (∼200 nm) of conventional microscopy. Here, we applied diffraction-unlimited, direct stochastic optical-reconstruction microscopy ( dSTORM) with a lateral resolution of ∼20 nm to analyze the focal nanostructure of the DSB marker histone γH2AX and the DNA-repair protein kinase (DNA-PK) in irradiated glioblastoma multiforme cells. Although standard confocal microscopy revealed substantial colocalization of immunostained γH2AX and DNA-PK, in our dSTORM images, the 2 proteins showed very little (if any) colocalization despite their close spatial proximity. We also found that γH2AX foci consisted of distinct circular subunits ("nanofoci") with a diameter of ∼45 nm, whereas DNA-PK displayed a diffuse, intrafocal distribution. We conclude that γH2AX nanofoci represent the elementary, structural units of DSB repair foci, that is, individual γH2AX-containing nucleosomes. dSTORM-based γH2AX nanofoci counting and distance measurements between nanofoci provided quantitative information on the total amount of chromatin involved in DSB repair as well as on the number and longitudinal distribution of γH2AX-containing nucleosomes in a chromatin fiber. We thus estimate that a single focus involves between ∼0.6 and ∼1.1 Mbp of chromatin, depending on radiation treatment. Because of their ability to unravel the nanostructure of DSB-repair foci, dSTORM and related single-molecule localization nanoscopy methods will likely emerge as powerful tools in biology and medicine to elucidate the effects of DNA damaging agents in cells.-Sisario, D., Memmel, S., Doose, S., Neubauer, J., Zimmermann, H., Flentje, M., Djuzenova, C. S., Sauer, M., Sukhorukov, V. L. Nanostructure of DNA repair foci revealed by superresolution microscopy.
电离辐射诱导DNA双链断裂（DSB）会导致微米级DNA修复灶的形成，由于传统显微镜的衍射极限（约200纳米），其纳米级别的组织结构仍不为人知。在此，我们应用横向分辨率约为20纳米的无衍射直接随机光学重建显微镜（dSTORM），来分析多形性胶质母细胞瘤细胞经照射后DSB标记物组蛋白γH2AX和DNA修复蛋白激酶（DNA-PK）的灶状纳米结构。尽管标准共聚焦显微镜显示免疫染色的γH2AX和DNA-PK有大量共定位，但在我们的dSTORM图像中，这两种蛋白质尽管空间距离很近，但共定位很少（如果有的话）。我们还发现，γH2AX灶由直径约45纳米的不同圆形亚基（“纳米灶”）组成，而DNA-PK呈现出弥散的、灶内分布。我们得出结论，γH2AX纳米灶代表DSB修复灶的基本结构单元，即单个含γH2AX的核小体。基于dSTORM的γH2AX纳米灶计数以及纳米灶之间的距离测量，提供了关于参与DSB修复的染色质总量以及染色质纤维中含γH2AX核小体的数量和纵向分布的定量信息。因此，我们估计单个灶涉及约0.6至1.1兆碱基对的染色质，这取决于辐射处理情况。由于能够揭示DSB修复灶的纳米结构，dSTORM及相关的单分子定位纳米显微镜方法可能会成为生物学和医学领域强大的工具，以阐明DNA损伤剂对细胞的影响。-西萨里奥，D.，梅梅尔，S.，杜泽，S.，诺伊鲍尔，J.，齐默尔曼，H.，弗伦特耶，M.，久泽诺娃，C. S.，绍尔，M.，苏霍鲁科夫，V. L. 超分辨率显微镜揭示的DNA修复灶的纳米结构。
