Quantification of DNA damage induced repair focus formation <i>via</i> super-resolution dSTORM localization microscopy

Varga, Dániel; Majoros, Hajnalka; Újfaludi, Zsuzsanna; Erdélyi, Miklós; Pankotai, Tibor

doi:10.1039/c9nr03696b

Cited by 29 publications

(30 citation statements)

References 66 publications

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“…Thus, future studies will be dedicated towards investigating the impact of 53BP1 oligomerization on DSB site chromatin structure, for example, by measurement of FRET between fluorescent histones 45 . Given the recent report that sub-micron domains within 53BP1 foci regulate chromatin topology 46 , it will also be important to place this dynamic picture of 53BP1 self-association in the context of a super-resolved image of 53BP1 foci formation 47 .…”

Section: Discussionmentioning

confidence: 99%

Spatiotemporal dynamics of 53BP1 dimer recruitment to a DNA double strand break

et al. 2020

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Tumor suppressor p53-binding protein 1 (53BP1) is a DNA repair protein essential for the detection, assessment, and resolution of DNA double strand breaks (DSBs). The presence of a DSB is signaled to 53BP1 via a local histone modification cascade that triggers the binding of 53BP1 dimers to chromatin flanking this type of lesion. While biochemical studies have established that 53BP1 exists as a dimer, it has never been shown in a living cell when or where 53BP1 dimerizes upon recruitment to a DSB site, or upon arrival at this nuclear location, how the DSB histone code to which 53BP1 dimers bind regulates retention and self-association into higher-order oligomers. Thus, here in live-cell nuclear architecture we quantify the spatiotemporal dynamics of 53BP1 oligomerization during a DSB DNA damage response by coupling fluorescence fluctuation spectroscopy (FFS) with the DSB inducible via AsiSI cell system (DIvA). From adopting this multiplexed approach, we find that preformed 53BP1 dimers relocate from the nucleoplasm to DSB sites, where consecutive recognition of ubiquitinated lysine 15 of histone 2A (H2AK15ub) and di-methylated lysine 20 of histone 4 (H4K20me2), leads to the assembly of 53BP1 oligomers and a mature 53BP1 foci structure.

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Section: Discussionmentioning

confidence: 99%

Spatiotemporal dynamics of 53BP1 dimer recruitment to a DNA double strand break

et al. 2020

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“…The reader may refer to the review by Schermelleh et al [102] for detailed information regarding SRM. Key SRM applications for DNA damage detection can be found in [103,104,105,106,107,108,109,110]. SRM resolution is ~100 nm axial and ~300 nm lateral, while it costs several hundreds of k USD.…”

Section: Detection Of Complex Dna Damagementioning

confidence: 99%

Ionizing Radiation and Complex DNA Damage: From Prediction to Detection Challenges and Biological Significance

Mavragani

Nikitaki

Kalospyros

et al. 2019

Cancers

143

107

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Biological responses to ionizing radiation (IR) have been studied for many years, generally showing the dependence of these responses on the quality of radiation, i.e., the radiation particle type and energy, types of DNA damage, dose and dose rate, type of cells, etc. There is accumulating evidence on the pivotal role of complex (clustered) DNA damage towards the determination of the final biological or even clinical outcome after exposure to IR. In this review, we provide literature evidence about the significant role of damage clustering and advancements that have been made through the years in its detection and prediction using Monte Carlo (MC) simulations. We conclude that in the future, emphasis should be given to a better understanding of the mechanistic links between the induction of complex DNA damage, its processing, and systemic effects at the organism level, like genomic instability and immune responses.

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“…After data analysis, artificial "pointillist" super-resolution images can be generated, in which the results of data analysis can additionally be encoded [40]. SMLM was applied to investigate the irradiation effects on chromatin nano-architecture [28,36,37] and protein organization at the nano scale [41][42][43], revealing systematic dose- [40,44] and locus-dependent [45] chromatin conformation changes. For multi-color localization microscopy of DNA damage markers along alpha tracks, we here applied molecular labelling with specific antibodies against γ-H2AX, MRE11, and 53BP1 in alpha-traversed cell nuclei and studied their distribution within γ-H2AX foci domains (super-foci) that demarcate the particle track.…”

Section: Introductionmentioning

confidence: 99%

Nanostructure of Clustered DNA Damage in Leukocytes after In-Solution Irradiation with the Alpha Emitter Ra-223

Scherthan

Lee

Maus

et al. 2019

Cancers

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Background: Cancer patients are increasingly treated with alpha-particle-emitting radiopharmaceuticals. At the subcellular level, alpha particles induce densely spaced ionizations and molecular damage. Induction of DNA lesions, especially clustered DNA double-strand breaks (DSBs), threatens a cell’s survival. Currently, it is under debate to what extent the spatial topology of the damaged chromatin regions and the repair protein arrangements are contributing. Methods: Super-resolution light microscopy (SMLM) in combination with cluster analysis of single molecule signal-point density regions of DSB repair markers was applied to investigate the nano-structure of DNA damage foci tracks of Ra-223 in-solution irradiated leukocytes. Results: Alpha-damaged chromatin tracks were efficiently outlined by γ-H2AX that formed large (super) foci composed of numerous 60–80 nm-sized nano-foci. Alpha damage tracks contained 60–70% of all γ-H2AX point signals in a nucleus, while less than 30% of 53BP1, MRE11 or p-ATM signals were located inside γ-H2AX damage tracks. MRE11 and p-ATM protein fluorescent tags formed focal nano-clusters of about 20 nm peak size. There were, on average, 12 (±9) MRE11 nanoclusters in a typical γ-H2AX-marked alpha track, suggesting a minimal number of MRE11-processed DSBs per track. Our SMLM data suggest regularly arranged nano-structures during DNA repair in the damaged chromatin domain.

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Quantification of DNA damage induced repair focus formation via super-resolution dSTORM localization microscopy

Abstract: A quantitative approach has been developed to analyse the morphology and distribution of DNA double-strand break induced single repair focus by super-resolution dSTORM microscopy.

Cited by 29 publications

References 66 publications

Spatiotemporal dynamics of 53BP1 dimer recruitment to a DNA double strand break

Spatiotemporal dynamics of 53BP1 dimer recruitment to a DNA double strand break

Ionizing Radiation and Complex DNA Damage: From Prediction to Detection Challenges and Biological Significance

Nanostructure of Clustered DNA Damage in Leukocytes after In-Solution Irradiation with the Alpha Emitter Ra-223

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