%0 Journal Article %1 sisario2018nanostructure %A Sisario, Dmitri %A Memmel, Simon %A Doose, Sören %A Neubauer, Julia %A Zimmermann, Heiko %A Flentje, Michael %A Djuzenova, Cholpon S. %A Sauer, Markus %A Sukhorukov, Vladimir L. %B The FASEB Journal %D 2018 %I Federation of American Societies for Experimental Biology %J The FASEB Journal %K doose sauer vladimir %P fj.201701435-- %R 10.1096/fj.201701435 %T Nanostructure of DNA repair foci revealed by superresolution microscopy %U https://doi.org/10.1096/fj.201701435 %X 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 ?H2AX and the DNA-repair protein kinase (DNA-PK) in irradiated glioblastoma multiforme cells. Although standard confocal microscopy revealed substantial colocalization of immunostained ?H2AX 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 ?H2AX foci consisted of distinct circular subunits (?nanofoci?) with a diameter of ?45 nm, whereas DNA-PK displayed a diffuse, intrafocal distribution. We conclude that ?H2AX nanofoci represent the elementary, structural units of DSB repair foci, that is, individual ?H2AX-containing nucleosomes. dSTORM-based ?H2AX 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 ?H2AX-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.

@article{sisario2018nanostructure, abstract = {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 ?H2AX and the DNA-repair protein kinase (DNA-PK) in irradiated glioblastoma multiforme cells. Although standard confocal microscopy revealed substantial colocalization of immunostained ?H2AX 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 ?H2AX foci consisted of distinct circular subunits (?nanofoci?) with a diameter of ?45 nm, whereas DNA-PK displayed a diffuse, intrafocal distribution. We conclude that ?H2AX nanofoci represent the elementary, structural units of DSB repair foci, that is, individual ?H2AX-containing nucleosomes. dSTORM-based ?H2AX 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 ?H2AX-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.}, added-at = {2018-06-14T10:46:53.000+0200}, author = {Sisario, Dmitri and Memmel, Simon and Doose, Sören and Neubauer, Julia and Zimmermann, Heiko and Flentje, Michael and Djuzenova, Cholpon S. and Sauer, Markus and Sukhorukov, Vladimir L.}, biburl = {https://www.bibsonomy.org/bibtex/2068e24c7e5d983316f989cf4cc023c75/reichert}, booktitle = {The FASEB Journal}, comment = {doi: 10.1096/fj.201701435}, doi = {10.1096/fj.201701435}, interhash = {d1dc3936daa871b7f100ab6d8090aee0}, intrahash = {068e24c7e5d983316f989cf4cc023c75}, issn = {08926638}, journal = {The FASEB Journal}, keywords = {doose sauer vladimir}, month = jun, pages = {fj.201701435--}, publisher = {Federation of American Societies for Experimental Biology}, timestamp = {2018-06-14T10:46:53.000+0200}, title = {Nanostructure of DNA repair foci revealed by superresolution microscopy}, url = {https://doi.org/10.1096/fj.201701435}, year = 2018 }