Identification of the elementary structural units of the DNA damage response

Natale, Francesco; Rapp, Alexander; Yu, Wei; Maiser, Andreas; Harz, Hartmann; Schöll, Armin; Grulich, Stephan; Anton, Tobias; Hörl, David; Chen, Wei; Durante, Marco; Taucher-Scholz, G.; Leonhardt, Heinrich; Cardoso, M. Cristina

doi:10.1038/ncomms15760

Cited by 160 publications

(202 citation statements)

References 51 publications

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“…Our finding that conventionally detected gH2AX foci consist of discrete subunits is further corroborated by the results of recent studies employing superresolution microscopy (13,15). The main achievement of our study is the direct visualization of previously unobserved gH2AXdecorated nucleosomes as elementary structural units of DSB repair foci.…”

Section: Discussionsupporting

confidence: 90%

“…Despite numerous studies regarding the involvement of gH2AX in DNA repair, the nanoscale organization of gH2AX and DDR proteins within IR-induced foci remains largely unknown because of the resolution limit of conventional fluorescence microscopy. At present, there are only a few reported studies using superresolution fluorescence microscopy to explore the nanoscale organization of DSB repair foci (13)(14)(15)(16). Those studies revealed that DSB foci consist of several subfoci with a lateral diameter ranging between ;100 and 200 nm.…”

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confidence: 99%

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Nanostructure of DNA repair foci revealed by superresolution microscopy

Sisario¹,

Memmel²,

Doose³

et al. 2018

The FASEB Journal

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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.

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Section: Discussionsupporting

confidence: 90%

mentioning

confidence: 99%

Nanostructure of DNA repair foci revealed by superresolution microscopy

Sisario¹,

Memmel²,

Doose³

et al. 2018

The FASEB Journal

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“…It has been shown that such foci can be separated into functionally relevant sub-foci or clusters of about the same size independent from the dose applied [23,28,29]. This has also been found for the cell nuclei analyzed here (see Figure S2).…”

Section: Resultssupporting

confidence: 78%

“…These clusters are not identical with γH2AX foci obtained by diffraction limited wide-field imaging. It has been shown that the foci are sub-divided into several sub-units which are compatible to the clusters described here [23,28]. Furthermore, the two parameters for the cluster recognition have been varied in order to ensure that the results of the subsequent analysis discussed in the following are not crucially dependent on this choice of parameters.…”

Section: Resultsmentioning

confidence: 93%

Using Persistent Homology as a New Approach for Super-Resolution Localization Microscopy Data Analysis and Classification of γH2AX Foci/Clusters

Hofmann

Krufczik

Heermann

et al. 2018

IJMS

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DNA double strand breaks (DSB) are the most severe damages in chromatin induced by ionizing radiation. In response to such environmentally determined stress situations, cells have developed repair mechanisms. Although many investigations have contributed to a detailed understanding of repair processes, e.g., homologous recombination repair or non-homologous end-joining, the question is not sufficiently answered, how a cell decides to apply a certain repair process at a certain damage site, since all different repair pathways could simultaneously occur in the same cell nucleus. One of the first processes after DSB induction is phosphorylation of the histone variant H2AX to γH2AX in the given surroundings of the damaged locus. Since the spatial organization of chromatin is not random, it may be conclusive that the spatial organization of γH2AX foci is also not random, and rather, contributes to accessibility of special repair proteins to the damaged site, and thus, to the following repair pathway at this given site. The aim of this article is to demonstrate a new approach to analyze repair foci by their topology in order to obtain a cell independent method of categorization. During the last decade, novel super-resolution fluorescence light microscopic techniques have enabled new insights into genome structure and spatial organization on the nano-scale in the order of 10 nm. One of these techniques is single molecule localization microscopy (SMLM) with which the spatial coordinates of single fluorescence molecules can precisely be determined and density and distance distributions can be calculated. This method is an appropriate tool to quantify complex changes of chromatin and to describe repair foci on the single molecule level. Based on the pointillist information obtained by SMLM from specifically labeled heterochromatin and γH2AX foci reflecting the chromatin morphology and repair foci topology, we have developed a new analytical methodology of foci or foci cluster characterization, respectively, by means of persistence homology. This method allows, for the first time, a cell independent comparison of two point distributions (here the point distributions of two γH2AX clusters) with each other of a selected ensample and to give a mathematical measure of their similarity. In order to demonstrate the feasibility of this approach, cells were irradiated by low LET (linear energy transfer) radiation with different doses and the heterochromatin and γH2AX foci were fluorescently labeled by antibodies for SMLM. By means of our new analysis method, we were able to show that the topology of clusters of γH2AX foci can be categorized depending on the distance to heterochromatin. This method opens up new possibilities to categorize spatial organization of point patterns by parameterization of topological similarity.

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“…There are no data in this regard and this question is subject for future consideration. Recent observations [25] shed new light on the mechanisms of DSB and H2AX foci induction and repair within TADs. In the present model TADs participate in exchange aberrations as structural blocks within chromosome of increased frequency of contacts of 100 kbp bins or chromosomal elements.…”

Section: Discussionmentioning

confidence: 99%

Spatial organization of the mouse chromosomes influences the landscape of intrachromosomal exchange aberration breakpoints

Eidelman

Salnikov

Slanina

et al. 2019

Preprint

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How 3D chromosome organization affects chromosomal aberrations is an important unresolved question in cell and radiation biology. In interphase the chromosomes form territories where chromatin folds into quite heterogeneous states. The mechanisms determining the spectrum of chromosome conformations remain poorly understood. We introduced the polymer model of mouse chromosome and generated the ensemble of 3D conformations. The chromosome model was validated against independent Hi-C (high-throughput chromosome conformation capture) data. The model described well Hi-C contact heatmap for chromosome 18 in pro-B mouse cells, both ATM-deficient and wild-type. We used the chromosome model to assess the role of chromosome structure in breakpoint distribution for intrachromosomal exchange aberrations. We investigated the effect of elevated frequency of breakpoints outside of the region of enzymatic breaksite. Chromosome aberration model explained breakpoint distribution under recurrent and ionizing radiation-induced DNA double-strand breaks in mouse chromosome 18 on the basis of contact-first mechanism. Overall, our results provide a framework for assessment of role of chromosome 3D organization on chromosome aberrations following DNA damage of different origin.

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Identification of the elementary structural units of the DNA damage response

Cited by 160 publications

References 51 publications

Nanostructure of DNA repair foci revealed by superresolution microscopy

Nanostructure of DNA repair foci revealed by superresolution microscopy

Using Persistent Homology as a New Approach for Super-Resolution Localization Microscopy Data Analysis and Classification of γH2AX Foci/Clusters

Spatial organization of the mouse chromosomes influences the landscape of intrachromosomal exchange aberration breakpoints

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