scholarly journals A damaged genome’s transcriptional landscape through multilayered expression profiling around in situ-mapped DNA double-strand breaks

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Fabio Iannelli ◽  
Alessandro Galbiati ◽  
Ilaria Capozzo ◽  
Quan Nguyen ◽  
Brian Magnuson ◽  
...  
Keyword(s):  
Expression Profiling ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Transcriptional Landscape

scholarly journals In situ Detection of Specific DNA Double Strand Breaks using Rolling Circle Amplification

Cell Cycle ◽  
2005 ◽  
Vol 4 (12) ◽  
pp. 1767-1773 ◽  
Author(s):  
Jia Li ◽  
C.S.H. Young ◽  
Paul M. Lizardia ◽  
David F. Stern
Keyword(s):  
Rolling Circle Amplification ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Rolling Circle ◽  
Strand Breaks ◽  
In Situ Detection

scholarly journals Transient Global Ischemia Triggers Expression of the DNA Damage-Inducible Gene GADD45 in the Rat Brain

1998 ◽  
Vol 18 (6) ◽  
pp. 646-657 ◽  
Author(s):  
Jun Chen ◽  
Koichi Uchimura ◽  
R. Anne Stetler ◽  
Raymond L. Zhu ◽  
Masaki Nakayama ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Blot Analysis ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Transient Ischemia ◽  
Strand Breaks ◽  
Transient Global Ischemia ◽  
Inducible Gene

Using in situ hybridization, Northern blot analysis, Western blot analysis, and immunocytochemistry, mRNA and protein expression of the novel DNA damage-inducible gene GADD45 was examined in the rat brain at 0.5, 2, 4, 8, 16, 24, 48, and 72 hours after 15 minutes of transient global ischemia. Transient ischemia produced by the four-vessel occlusion method resulted in DNA double-strand breaks and delayed neuronal cell death in vulnerable neurons of the hippocampal CA1 sector, the hilus, dorsal caudate-putamen, and thalamus, as shown by in situ DNA nick end-labeling and histologic staining. GADD45 mRNA was transiently increased in less-vulnerable regions such as the parietal cortex (up to 8 hours after ischemia) and dentate granule cells (up to 24 hours after ischemia) but was persistently increased in vulnerable neurons such as CA1 pyramidal neurons (up to 48 hours). GADD45 immunoreactivity was increased in both vulnerable and less-vulnerable regions at earlier reperfusion periods (4 to 16 hours), but thereafter immunoreactivity was decreased below control levels in most vulnerable regions before delayed cell death and DNA double-strand breaks. At 72 hours after transient ischemia, a moderate increase in GADD45 immunoreactivity was still detectable in some CA3 neurons and in a few surviving neurons in the CA1 region. Double staining performed at 16 to 72 hours after ischemia revealed that GADD45 immunoreactivity was persistently increased in neurons that did not develop DNA damage. Because GADD45 protein may participate in the DNA excision repair process and because it has been shown that this protein is also overexpressed in neurons that survive focal ischemia and kainate-induced epileptic seizures, the results reported here support the hypothesis that GADD45 could have a protective role in neuronal injury.

Induction of in situ DNA double-strand breaks and apoptosis by 200 MeV protons and 10 MV X-rays in human tumour cell lines

2010 ◽  
Vol 87 (1) ◽  
pp. 57-70 ◽  
Author(s):  
Ariungerel Gerelchuluun ◽  
Zhengshan Hong ◽  
Lue Sun ◽  
Kenshi Suzuki ◽  
Toshiyuki Terunuma ◽  
...  
Keyword(s):  
Tumour Cell ◽  
Double Strand Breaks ◽  
X Rays ◽  
Dna Double Strand Breaks ◽  
Human Tumour ◽  
Strand Breaks ◽  
Tumour Cell Lines ◽  
Human Tumour Cell Lines ◽  
Mev Protons

scholarly journals Cell Type–Specific Quantification of Telomere Length and DNA Double-strand Breaks in Individual Lung Cells by Fluorescence In Situ Hybridization and Fluorescent Immunohistochemistry

2018 ◽  
Vol 66 (7) ◽  
pp. 485-495 ◽  
Author(s):  
Aernoud A. van Batenburg ◽  
Karin M. Kazemier ◽  
Ton Peeters ◽  
Matthijs F. M. van Oosterhout ◽  
Joanne J. van der Vis ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Telomere Length ◽  
Dna Sequences ◽  
Double Strand Breaks ◽  
Telomere Maintenance ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

Telomeres are small repetitive DNA sequences at the ends of chromosomes which act as a buffer in age-dependent DNA shortening. Insufficient telomere repeats will be recognized as double-strand breaks. Presently, it is becoming more evident that telomere attrition, whether or not caused by mutations in telomere maintenance genes, plays an important role in many inflammatory and age-associated diseases. In this report, a method to (semi)quantitatively assess telomere length and DNA double-strand breaks in formalin-fixed paraffin-embedded (FFPE) tissue is described. Therefore, a novel combination of quantitative fluorescence in situ hybridization, tissue elution, and immunofluorescence staining techniques was developed. Caveolin-1 (type 1 pneumocytes), pro-surfactant protein C (type 2 pneumocytes), club cell-10 (club cells), and alpha smooth muscle actin (smooth muscle cells) markers were used to identify cell types. To visualize all the different probes, restaining the tissue by heat-mediated slide elution is essential. Fluorescent signals of telomeres and DNA double-strand breaks were quantified using the Telometer plugin of ImageJ. As example, we analyzed lung tissue from a familial pulmonary fibrosis patient with a mutation in the telomere-associated gene poly(A)-specific ribonuclease ( PARN). The protocol displays a novel opportunity to directly quantitatively link DNA double-strand breaks to telomere length in specific FFPE cells.

CBIO-08. ENDOGENOUS DNA DOUBLE STRAND BREAKS ACTIVATE HETEROGENOUS DNA DAMAGE SIGNALING IN IDH1/2 MUTANT GLIOMAS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi28-vi29
Author(s):  
Gaspar Kitange ◽  
Rachael Vaubel ◽  
Jann Sarkaria
Keyword(s):  
Dna Damage ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Isocitrate Dehydrogenase 1 ◽  
Strand Breaks ◽  
Dna Damage Signaling ◽  
Alternative Lengthening ◽  
Systematic Effort ◽  
Patient Specimen

Abstract Isocitrate dehydrogenase 1/2 (IDH1/2) mutations are common in astrocytic glioma and are frequently coupled with TP53 and ATRX mutations. Collectively, these alterations cause genomic instability leading to high basal DNA double strand breaks (DSBs). Understanding how IDH/TP53/ATRX mutant cells process endogenous DSBs may help exploit inhibitors of DNA damage response (DDR) for the treatment of patients with IDH mutant gliomas. Through systematic effort to uncover the mechanisms involved in repair of endogenous DSBs in IDH1/2 mutant GBMs, we have discovered that high basal phosphorylated DNA-PK (p-DNA-PK) was characteristic of an IDH1/TP53/ATRX mutant GBM164 patient derived xenograft (PDX) but not in another IDH1 mutant GBM196 PDX. Immunofluorescence (IF) studies in patient specimen from which GBM164 was derived showed that p-DNA-PK co-localized with g-H2AX, 53BP1 or H4K20me2 (but not p-RPA) the known surrogates of DSBs. In contrast, p-DNA-PK was absent in the patient specimen from which GBM196 was derived, which otherwise had equally intense g-H2AX immunostaining colocalized with p-RPA. An independent IF study involving 11 IDH1 wild-type (WT) and 11 IDH1 mutant GBM patient samples, the p-DNA-PK was observed in 3 (27%) of 11 IDH1 mutant samples while IDH1 WT tumors were negative for p-DNA-PK. A telomere specific fluorescence in situ hybridization (Tel-FISH) confirmed elevated alternative lengthening of telomere (ALT) activity in GBM196 (but not in GBM164) indicative of HR proficiency. Consistently, HR related genes, including BRCA1 and MRE11A, were found upregulated in ALT-positive GBM196 as compared to those in GBM164. Interestingly, ALT+ GBM196 cells were highly vulnerable to inhibitors of ATM and ATR pathways. In conclusion, IDH1/TP53/ATRX mutant gliomas can be subdivided into HR-mediated ALT-positive group, which repairs the endogenous DSBs by HR (e.g. GBM196) and an ALT-negative/p-DNA-PK group, which repairs DSBs by c-NHEJ (e.g. GBM164) and this subdivision can be developed as a prescient biomarker of sensitivity to DDR inhibitors.

scholarly journals Breaks Labeling in situ and sequencing (BLISS)

2019 ◽  
Author(s):  
Winston X. Yan ◽  
Reza Mirzazadeh ◽  
Silvano Garnerone ◽  
David Scott ◽  
Martin W. Schneider ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Low Input ◽  
Tissue Sections ◽  
Genome Wide ◽  
Transcription 3 ◽  
Target Activity

Abstract Precisely measuring the location and frequency of DNA double-strand breaks \(DSBs) along the genome is instrumental to understanding genomic fragility, but current methods are limited in versatility, sensitivity, or practicality. Here, we present Breaks Labeling _In Situ_ and Sequencing \(BLISS), featuring: 1) direct labeling of DSBs in fixed cells or tissue sections on a solid surface; 2) low-input requirement by linear amplification of tagged DSBs by _in vitro_ transcription; 3) quantification of DSBs through unique molecular identifiers; and 4) easy scalability and multiplexing. We apply BLISS to profile endogenous and exogenous DSBs in low-input samples of cancer cells, embryonic stem cells, and liver tissue. We demonstrate the sensitivity of BLISS by assessing the genome-wide off-target activity of two CRISPR-associated RNA-guided endonucleases, Cas9 and Cpf1, observing that Cpf1 has higher specificity than Cas9. Our results establish BLISS as a versatile, sensitive, and efficient method for genome-wide DSB mapping in many applications. W.Yan and R.Mirzazadeh equally contributed to this work.

Repair pathway choice for double-strand breaks

2020 ◽  
Vol 64 (5) ◽  
pp. 765-777 ◽  
Author(s):  
Yixi Xu ◽  
Dongyi Xu
Keyword(s):  
Cell Cycle ◽  
Double Strand Breaks ◽  
Homologous Region ◽  
Gene Repair ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
Environmental Radiation ◽  
Strand Breaks ◽  
Dna End Resection ◽  
End Resection

Abstract Deoxyribonucleic acid (DNA) is at a constant risk of damage from endogenous substances, environmental radiation, and chemical stressors. DNA double-strand breaks (DSBs) pose a significant threat to genomic integrity and cell survival. There are two major pathways for DSB repair: nonhomologous end-joining (NHEJ) and homologous recombination (HR). The extent of DNA end resection, which determines the length of the 3′ single-stranded DNA (ssDNA) overhang, is the primary factor that determines whether repair is carried out via NHEJ or HR. NHEJ, which does not require a 3′ ssDNA tail, occurs throughout the cell cycle. 53BP1 and the cofactors PTIP or RIF1-shieldin protect the broken DNA end, inhibit long-range end resection and thus promote NHEJ. In contrast, HR mainly occurs during the S/G2 phase and requires DNA end processing to create a 3′ tail that can invade a homologous region, ensuring faithful gene repair. BRCA1 and the cofactors CtIP, EXO1, BLM/DNA2, and the MRE11–RAD50–NBS1 (MRN) complex promote DNA end resection and thus HR. DNA resection is influenced by the cell cycle, the chromatin environment, and the complexity of the DNA end break. Herein, we summarize the key factors involved in repair pathway selection for DSBs and discuss recent related publications.

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