Superresolution light microscopy shows nanostructure of carbon ion radiation‐induced DNA double‐strand break repair foci

Perez, Ramon Lopez; Best, Gerrit; Nicolay, Nils H.; Greubel, C.; Roßberger, Sabrina; Reindl, Judith; Dollinger, G.; Weber, Klaus; Cremer, Christoph; Huber, Peter E.

doi:10.1096/fj.201500106r

Cited by 43 publications

(31 citation statements)

References 70 publications

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“…The γH2AX nano-foci we identified are two- to threefold smaller—with lateral diameters of ∼200 nm—and contain ∼10% of the conventionally estimated Mbp DNA content42. Similar γH2AX substructures sizes were recently measured after heavy ion irradiation43, despite the highly ionizing power charged particles possess, thus further supporting our findings.…”

Section: Discussionsupporting

confidence: 89%

Identification of the elementary structural units of the DNA damage response

et al. 2017

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Histone H2AX phosphorylation is an early signalling event triggered by DNA double-strand breaks (DSBs). To elucidate the elementary units of phospho-H2AX-labelled chromatin, we integrate super-resolution microscopy of phospho-H2AX during DNA repair in human cells with genome-wide sequencing analyses. Here we identify phospho-H2AX chromatin domains in the nanometre range with median length of ∼75 kb. Correlation analysis with over 60 genomic features shows a time-dependent euchromatin-to-heterochromatin repair trend. After X-ray or CRISPR-Cas9-mediated DSBs, phospho-H2AX-labelled heterochromatin exhibits DNA decondensation while retaining heterochromatic histone marks, indicating that chromatin structural and molecular determinants are uncoupled during repair. The phospho-H2AX nano-domains arrange into higher-order clustered structures of discontinuously phosphorylated chromatin, flanked by CTCF. CTCF knockdown impairs spreading of the phosphorylation throughout the 3D-looped nano-domains. Co-staining of phospho-H2AX with phospho-Ku70 and TUNEL reveals that clusters rather than nano-foci represent single DSBs. Hence, each chromatin loop is a nano-focus, whose clusters correspond to previously known phospho-H2AX foci.

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Section: Discussionsupporting

confidence: 89%

Identification of the elementary structural units of the DNA damage response

et al. 2017

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“…61‐64 In the case of ∼ 1‐2 high-LET iron ion traversals, nearly all of the energy deposited by their tracks and associated δ-ray penumbras involve more limited nuclear volumes and genomic regions located directly along the particle track 65,66 with much higher local ionization densities and resulting DSBs and clustered lesions. 67 HZE ion-induced DSBs have been shown to be more refractory and slower to repair than low LET IR-induced DSBs, with a greater proportion being preferentially repaired by Rad51-mediated homologous recombinational repair (HRR), 68,69 yielding much higher levels of simple and complex chromosomal rearrangements post irradiation. 70 Interestingly, two recent studies demonstrate that HSC have reduced DSB repair capacities compared with mature T-cells.…”

Section: Discussionmentioning

confidence: 99%

In vitro and in vivo assessment of direct effects of simulated solar and galactic cosmic radiation on human hematopoietic stem/progenitor cells

et al. 2016

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Future deep space missions to Mars and near-Earth asteroids will expose astronauts to chronic solar energetic particles (SEP) and galactic cosmic ray (GCR) radiation, and likely one or more solar particle events (SPEs). Given the inherent radiosensitivity of hematopoietic cells and short latency period of leukemias, space radiation-induced hematopoietic damage poses a particular threat to astronauts on extended missions. We show that exposing human hematopoietic stem/progenitor cells (HSC) to extended mission-relevant doses of accelerated high-energy protons and iron ions leads to the following: (1) introduces mutations that are frequently located within genes involved in hematopoiesis and are distinct from those induced by γ-radiation; (2) markedly reduces in vitro colony formation; (3) markedly alters engraftment and lineage commitment in vivo; and (4) leads to the development, in vivo, of what appears to be T-ALL. Sequential exposure to protons and iron ions (as typically occurs in deep space) proved far more deleterious to HSC genome integrity and function than either particle species alone. Our results represent a critical step for more accurately estimating risks to the human hematopoietic system from space radiation, identifying and better defining molecular mechanisms by which space radiation impairs hematopoiesis and induces leukemogenesis, as well as for developing appropriately targeted countermeasures.

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“…Recent work analyzed the clustering of DSB repair factors in detail using high resolution microscopy101112 and nanoscopy1113141516 in combination with state of the art correlation and clustering analysis methods. With these methods it is possible to gain a deeper understanding of the functionality of DSB repair proteins and their interactions.…”

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confidence: 99%

Chromatin organization revealed by nanostructure of irradiation induced γH2AX, 53BP1 and Rad51 foci

Reindl

Girst

Walsh

et al. 2017

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The spatial distribution of DSB repair factors γH2AX, 53BP1 and Rad51 in ionizing radiation induced foci (IRIF) in HeLa cells using super resolution STED nanoscopy after low and high linear energy transfer (LET) irradiation was investigated. 53BP1 and γH2AX form IRIF with same mean size of (540 ± 40) nm after high LET irradiation while the size after low LET irradiation is significantly smaller. The IRIF of both repair factors show nanostructures with partial anti-correlation. These structures are related to domains formed within the chromatin territories marked by γH2AX while 53BP1 is mainly situated in the perichromatin region. The nanostructures have a mean size of (129 ± 6) nm and are found to be irrespective of the applied LET and the labelled damage marker. In contrast, Rad51 shows no nanostructure and a mean size of (143 ± 13) nm independent of LET. Although Rad51 is surrounded by 53BP1 it strongly anti-correlates meaning an exclusion of 53BP1 next to DSB when decision for homologous DSB repair happened.Ionizing radiation induces a variety of different types of damage when targeted to living cells. Severe damage, which can influence cell survival or lead to carcinogenesis, occurs due to ionizing events in the DNA molecule itself. The most lethal of these types of DNA damages are the double-strand breaks (DSB), as they may lead to genetic alterations which in turn can be responsible for cell death or carcigonesis. Mammalian cells react with a variety of complex response mechanisms to DSB induction. One main reaction is the phosphorylation of the histone variant H2AX at serine 139 (S139) to obtain γ H2AX through kinases such as ATM, ATR and DNA-PK 1 . The γ H2AX domains occur in mega-base-pair (Mbp) large regions of the chromatin around DSB 2-6 and can be visualized as so-called ionizing radiation induced foci (IRIF) 7 . The recruitment and activation of proteins due to damage induction can later on lead to the repair of DSB. The cell has different repair mechanisms to properly rejoin the ends of a DSB, including the possibly error-prone non-homologous end joining (NHEJ) 8 and the in most cases error-free homologous recombination (HR) 9 . HR is limited to the S/G2 cell cycle phase, due to the fact that a homologous sister chromatin is needed in close vicinity to the DSB as a template to repair the damaged chromatin 2-4 . As a backup pathway for failed NHEJ in G1 an alternative end-joining pathway (alt-EJ) has previously been identified, which works as a last resort, when the other pathways fail 8 . Recent work analyzed the clustering of DSB repair factors in detail using high resolution microscopy 10-12 and nanoscopy 11,[13][14][15][16] in combination with state of the art correlation and clustering analysis methods. With these methods it is possible to gain a deeper understanding of the functionality of DSB repair proteins and their interactions. After the first reactions to DSB induction, such as phosphorylation of H2AX (γ H2AX), the recruitment of downstream repair proteins starts for NHEJ as well as f...

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Superresolution light microscopy shows nanostructure of carbon ion radiation‐induced DNA double‐strand break repair foci

Cited by 43 publications

References 70 publications

Identification of the elementary structural units of the DNA damage response

Identification of the elementary structural units of the DNA damage response

In vitro and in vivo assessment of direct effects of simulated solar and galactic cosmic radiation on human hematopoietic stem/progenitor cells

Chromatin organization revealed by nanostructure of irradiation induced γH2AX, 53BP1 and Rad51 foci

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