Microcollimator for Micrometer-Wide Stripe Irradiation of Cells Using 20–30 keV X Rays

Pataky, Kristopher; Villanueva, G.; Liani, André; Zgheib, Omar; Jenkins, Nathan T.; Halazonetis, Demetrios J.; Halazonetis, Thanos D.; Brugger, Juergen

doi:10.1667/rr1483.1

Cited by 6 publications

(5 citation statements)

References 26 publications

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“…Finally, we utilized a newly developed microcollimator for X rays (28) to document that the IR-induced foci of the GFP-53BP1 fusion proteins indeed represent sites of DNA DSBs. IR-induced foci of endogenous 53BP1 were originally considered to represent sites of DNA DSBs on the basis of their colocalization with foci of phosphorylated histone H2AX and foci of NBS1 (4,30,33,44).…”

Section: Discussionmentioning

confidence: 99%

“…Stably transfected cells were cultured on 18-mm coverslips, which were then placed at the outlet of an X-ray microcollimator (28). The cells were exposed to IR using the X-Rad 320 irradiator operating at 30 kV and 25 mA.…”

Section: Methodsmentioning

confidence: 99%

“…Throughout this project, we have considered the ability of the various GFP-53BP1 fusion proteins to form IR-induced foci to be synonymous with recruitment to sites of DNA DSBs. To verify that this is indeed the case, we developed an X-ray collimator that can limit the irradiated field to areas whose smallest dimension is in the micrometer range (28). This microcollimator consists of silicon plates with dimensions of 1 cm by 2 cm by 380 m. On their surfaces the silicon plates have grooves that are 1 m deep and run the entire length of the plate (Fig.…”

Section: Vol 29 2009 Recognition Of Dna Double-strand Breaks By 53bmentioning

confidence: 99%

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An Oligomerized 53BP1 Tudor Domain Suffices for Recognition of DNA Double-Strand Breaks

Zgheib

Pataky

Brugger

et al. 2009

Molecular and Cellular Biology

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111

117

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53BP1, the vertebrate ortholog of the budding yeast Rad9 and fission yeast Crb2/Rhp9 checkpoint proteins, is recruited rapidly to sites of DNA double-strand breaks (DSBs). A tandem tudor domain in human 53BP1 that recognizes methylated residues in the histone core is necessary, but not sufficient, for efficient recruitment. By analysis of deletion mutants, we identify here additional elements in 53BP1 that facilitate recognition of DNA DSBs. The first element corresponds to an independently folding oligomerization domain. Replacement of this domain with heterologous tetramerization domains preserves the ability of 53BP1 to recognize DNA DSBs. A second element is only about 15 amino acids long and appears to be a C-terminal extension of the tudor domain, rather than an independently functioning domain. Recruitment of 53BP1 to sites of DNA DSBs is facilitated by histone H2AX phosphorylation and ubiquitination. However, none of the 53BP1 domains/ elements important for recruitment are known to bind phosphopeptides or ubiquitin, suggesting that histone phosphorylation and ubiquitination regulate 53BP1 recruitment to sites of DNA DSBs indirectly.Monitoring the presence of DNA double-strand breaks (DSBs) is critical for maintaining genomic stability. In eukaryotes, the DNA DSB checkpoint pathway senses the presence of DNA DSBs and activates effectors that induce cell cycle arrest, apoptosis, or senescence. Key components of this pathway in human cells are DNA DSB sensors such as 53BP1 and the Mre11-Rad50-NBS1 complex, the signal transducing kinase ATM, and effectors downstream of ATM such as the kinase Chk2 and the transcription factor p53 (1,16,25).53BP1 is one of the DNA damage response proteins that is recruited very efficiently to sites of DNA DSBs. Its recruitment can be visualized either by immunofluorescence of fixed cells or by monitoring live cells expressing 53BP1 fused to green fluorescent protein (GFP). In cells exposed to ionizing radiation (IR), the recruitment of 53BP1 to sites of DNA DSBs becomes evident by its localization to foci that are distributed throughout the nucleus; these foci are thought to correspond to sites of DNA DSBs (4,20,30,33,44). When DNA damage is induced in specific subnuclear compartments, for example, by UV laser light or by highly charged energetic particles, then 53BP1 localizes to the subnuclear compartments, where the DNA damage was induced (5, 8).The ability to easily monitor recruitment of 53BP1 to sites of DNA DSBs has allowed significant progress to be made regarding how this protein recognizes DNA damage. Mammalian 53BP1 and its orthologs Rad9 and Crb2/Rhp9, in budding and fission yeast, respectively, recognize DNA DSBs via a tandem tudor domain that binds to methylated histones (18, 31). Human 53BP1 recognizes either methylated K79 of histone H3 or methylated K20 of histone H4 (6,18,32,46), Rad9 recognizes exclusively methylated K79 of histone H3 (13, 43), and Crb2/Rhp9 recognizes exclusively methylated K20 of histone H4 (10, 31). Both K79 of histone H3 and K20 of hist...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Vol 29 2009 Recognition Of Dna Double-strand Breaks By 53bmentioning

confidence: 99%

See 1 more Smart Citation

An Oligomerized 53BP1 Tudor Domain Suffices for Recognition of DNA Double-Strand Breaks

Zgheib

Pataky

Brugger

et al. 2009

Molecular and Cellular Biology

Self Cite

111

117

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“…In addition to the use of endonucleases, several localized irradiation techniques have been developed to introduce discrete sites of DNA breaks in the cell nucleus, including partial cell volume irradiation with ultrasoft X-rays through a gridded shield (Nelms et al 1998) or highvoltage X-rays through a microcollimator (Pataky et al 2009). Similarly, local UV-C irradiation through micropore filters combined with the expression of UVDE (UV-specific endonuclease) in cells defective in repairing UV lesions has been used to generate localized SSBs (Okano et al 2003).…”

Section: Responses To Dna Breaks In a Physiological Contextmentioning

confidence: 99%

Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications

Polo¹,

Jackson²

2011

Genes Dev.

978

946

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Genome integrity is constantly monitored by sophisticated cellular networks, collectively termed the DNA damage response (DDR). A common feature of DDR proteins is their mobilization in response to genotoxic stress. Here, we outline how the development of various complementary methodologies has provided valuable insights into the spatiotemporal dynamics of DDR protein assembly/disassembly at sites of DNA strand breaks in eukaryotic cells. Considerable advances have also been made in understanding the underlying molecular mechanisms for these events, with post-translational modifications of DDR factors being shown to play prominent roles in controlling the formation of foci in response to DNA-damaging agents. We review these regulatory mechanisms and discuss their biological significance to the DDR.

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“…The efficiency of shielding was confirmed by Gafchromic film measurement with less than 2.3% of the dose delivered reaching under the shielded area and a sharp transition (dose falls from 90% to 10% within 2 mm). Additionally we used gallium arsenide microcollimator from MicroLeman (Lausanne, Switzerland) [30] , which allows exposure of samples with localized (1–10 µm wide stripes) oft 30 KVp X-rays ( Figure 1A ).…”

Section: Methodsmentioning

confidence: 99%

Mechanisms of DNA Damage Response to Targeted Irradiation in Organotypic 3D Skin Cultures

et al. 2014

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DNA damage (caused by direct cellular exposure and bystander signaling) and the complex pathways involved in its repair are critical events underpinning cellular and tissue response following radiation exposures. There are limited data addressing the dynamics of DNA damage induction and repair in the skin particularly in areas not directly exposed. Here we investigate the mechanisms regulating DNA damage, repair, intracellular signalling and their impact on premature differentiation and development of inflammatory-like response in the irradiated and surrounding areas of a 3D organotypic skin model. Following localized low-LET irradiation (225 kVp X-rays), low levels of 53BP1 foci were observed in the 3D model (3.8±0.28 foci/Gy/cell) with foci persisting and increasing in size up to 48 h post irradiation. In contrast, in cell monolayers 14.2±0.6 foci/Gy/cell and biphasic repair kinetics with repair completed before 24 h was observed. These differences are linked to differences in cellular status with variable level of p21 driving apoptotic signalling in 2D and accelerated differentiation in both the directly irradiated and bystander areas of the 3D model. The signalling pathways utilized by irradiated keratinocytes to induce DNA damage in non-exposed areas of the skin involved the NF-κB transcription factor and its downstream target COX-2.

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Microcollimator for Micrometer-Wide Stripe Irradiation of Cells Using 20–30 keV X Rays

Cited by 6 publications

References 26 publications

An Oligomerized 53BP1 Tudor Domain Suffices for Recognition of DNA Double-Strand Breaks

An Oligomerized 53BP1 Tudor Domain Suffices for Recognition of DNA Double-Strand Breaks

Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications

Mechanisms of DNA Damage Response to Targeted Irradiation in Organotypic 3D Skin Cultures

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