AN ImageJ-BASED ALGORITHM FOR A SEMI-AUTOMATED METHOD FOR MICROSCOPIC IMAGE ENHANCEMENT AND DNA REPAIR FOCI COUNTING

2015 ◽  
Vol 4 (1) ◽  
pp. 75-82
Author(s):  
Dmitry Klokov ◽  
Roopa Suppiah
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Low Dose ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Low Dose Radiation ◽  
Experimental Conditions ◽  
Strand Breaks ◽  
Automated Method ◽  
Dose Radiation

Proper evaluation of the health risks of low-dose ionizing radiation exposure heavily relies on the ability to accurately measure very low levels of DNA damage in cells. One of the most sensitive methods for measuring DNA damage levels is the quantification of DNA repair foci that consist of macromolecular aggregates of DNA repair proteins, such as γH2AX and 53BP1, forming around individual DNA double-strand breaks. They can be quantified using immunofluorescence microscopy and are widely used as markers of DNA double-strand breaks. However this quantification, if performed manually, may be very tedious and prone to inter-individual bias. Low-dose radiation studies are especially sensitive to this potential bias due to a very low magnitude of the effects anticipated. Therefore, we designed and validated an algorithm for the semi-automated processing of microscopic images and quantification of DNA repair foci. The algorithm uses ImageJ, a freely available image analysis software that is customizable to individual cellular properties or experimental conditions. We validated the algorithm using immunolabeled 53BP1 and γH2AX in normal human fibroblast AG01522 cells under both normal and irradiated conditions. This method is easy to learn, can be used by nontrained personnel, and can help avoiding discrepancies in inter-laboratory comparison studies examining the effects of low-dose radiation.

scholarly journals Recent advances in the nucleolar responses to DNA double-strand breaks

2020 ◽  
Vol 48 (17) ◽  
pp. 9449-9461
Author(s):  
Lea Milling Korsholm ◽  
Zita Gál ◽  
Blanca Nieto ◽  
Oliver Quevedo ◽  
Stavroula Boukoura ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Ribosomal Dna ◽  
Repetitive Sequences ◽  
Dna Lesions ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response

Abstract DNA damage poses a serious threat to human health and cells therefore continuously monitor and repair DNA lesions across the genome. Ribosomal DNA is a genomic domain that represents a particular challenge due to repetitive sequences, high transcriptional activity and its localization in the nucleolus, where the accessibility of DNA repair factors is limited. Recent discoveries have significantly extended our understanding of how cells respond to DNA double-strand breaks (DSBs) in the nucleolus, and new kinases and multiple down-stream targets have been identified. Restructuring of the nucleolus can occur as a consequence of DSBs and new data point to an active regulation of this process, challenging previous views. Furthermore, new insights into coordination of cell cycle phases and ribosomal DNA repair argue against existing concepts. In addition, the importance of nucleolar-DNA damage response (n-DDR) mechanisms for maintenance of genome stability and the potential of such factors as anti-cancer targets is becoming apparent. This review will provide a detailed discussion of recent findings and their implications for our understanding of the n-DDR. The n-DDR shares features with the DNA damage response (DDR) elsewhere in the genome but is also emerging as an independent response unique to ribosomal DNA and the nucleolus.

scholarly journals Screen identifies DYRK1B network as mediator of transcription repression on damaged chromatin

2020 ◽  
Vol 117 (29) ◽  
pp. 17019-17030 ◽  
Author(s):  
Chao Dong ◽  
Kirk L. West ◽  
Xin Yi Tan ◽  
Junshi Li ◽  
Toyotaka Ishibashi ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Gene Silencing ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Active Chromatin ◽  
Chemical Library ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Damage Response

DNA double-strand breaks (DSBs) trigger transient pausing of nearby transcription, an emerging ATM-dependent response that suppresses chromosomal instability. We screened a chemical library designed to target the human kinome for new activities that mediate gene silencing on DSB-flanking chromatin, and have uncovered the DYRK1B kinase as an early respondent to DNA damage. We showed that DYRK1B is swiftly and transiently recruited to laser-microirradiated sites, and that genetic inactivation of DYRK1B or its kinase activity attenuated DSB-induced gene silencing and led to compromised DNA repair. Notably, global transcription shutdown alleviated DNA repair defects associated with DYRK1B loss, suggesting that DYRK1B is strictly required for DSB repair on active chromatin. We also found that DYRK1B mediates transcription silencing in part via phosphorylating and enforcing DSB accumulation of the histone methyltransferase EHMT2. Together, our findings unveil the DYRK1B signaling network as a key branch of mammalian DNA damage response circuitries, and establish the DYRK1B–EHMT2 axis as an effector that coordinates DSB repair on transcribed chromatin.

scholarly journals Genomic Instability and Cancer Risk Associated with Erroneous DNA Repair

2021 ◽  
Vol 22 (22) ◽  
pp. 12254
Author(s):  
Ken-ichi Yoshioka ◽  
Rika Kusumoto-Matsuo ◽  
Yusuke Matsuno ◽  
Masamichi Ishiai
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Cancer Risk ◽  
Genomic Instability ◽  
Double Strand Breaks ◽  
Genomic Rearrangements ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
Brca1 And Brca2 ◽  
Strand Breaks

Many cancers develop as a consequence of genomic instability, which induces genomic rearrangements and nucleotide mutations. Failure to correct DNA damage in DNA repair defective cells, such as in BRCA1 and BRCA2 mutated backgrounds, is directly associated with increased cancer risk. Genomic rearrangement is generally a consequence of erroneous repair of DNA double-strand breaks (DSBs), though paradoxically, many cancers develop in the absence of DNA repair defects. DNA repair systems are essential for cell survival, and in cancers deficient in one repair pathway, other pathways can become upregulated. In this review, we examine the current literature on genomic alterations in cancer cells and the association between these alterations and DNA repair pathway inactivation and upregulation.

scholarly journals Vertebrate cells genetically deficient for Cdc14A or Cdc14B retain DNA damage checkpoint proficiency but are impaired in DNA repair

2010 ◽  
Vol 189 (4) ◽  
pp. 631-639 ◽  
Author(s):  
Annamaria Mocciaro ◽  
Eli Berdougo ◽  
Kang Zeng ◽  
Elizabeth Black ◽  
Paola Vagnarelli ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Retinal Pigment ◽  
Double Strand Breaks ◽  
Retinal Pigment Epithelial Cells ◽  
Dna Damage Checkpoint ◽  
Dna Double Strand Breaks ◽  
Central Function ◽  
Strand Breaks ◽  
Human Telomerase

A recent study suggested that human Cdc14B phosphatase has a central function in the G2 DNA damage checkpoint. In this study, we show that chicken DT40, human HCT116, and human telomerase reverse transcription–immortalized retinal pigment epithelial cells deleted for the Cdc14A or Cdc14B gene are DNA damage checkpoint proficient and arrest efficiently in G2 in response to irradiation. Cdc14A knockout (KO) or Cdc14B-KO cells also maintain normal levels of Chk1 and Chk2 activation after irradiation. Surprisingly, however, irradiation-induced γ-H2A.X foci and DNA double-strand breaks persist longer in Cdc14A-KO or Cdc14B-KO cells than controls, suggesting that Cdc14 phosphatases are required for efficient DNA repair.

Synthetic Lethality In CLL With DNA Damage Response Defect By Targeting ATR Pathway

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 120-120
Author(s):  
Tatjana Stankovic ◽  
Davies Nicholas ◽  
Marwan Kwok ◽  
Edward Smith ◽  
Eliot Yates ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Synthetic Lethality ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Single Strand Break ◽  
Strand Breaks ◽  
Damage Response ◽  
Repair Pathways

Abstract Ataxia Telangiectasia Mutated (ATM) protein coordinates responses to DNA double strand breaks (DSBs) and the ATM-null status caused by biallelic ATM gene inactivation in chronic lymphocytic leukemia (CLL) results in resistance to p53-dependent apoptosis. Accordingly, alternative strategies to target ATM-null CLL are needed. ATM is a serine/threonine protein kinase that synchronises rapid DNA damage response (DDR) to DNA double strand breaks (DSBs) with activation of cell cycle checkpoints, DNA repair and apoptosis via p53 activation. ATM-null cells are defective in a type of DSB repair that involves homologous recombination and rely on co-operating and compensatory DNA repair pathways for their survival. Therefore, inhibition of DNA repair pathways to which CLL cells with loss of ATM signalling become addicted could provide ‘synthetic lethality’ and induce tumour specific killing. Indeed, we have recently shown that inhibition of a single strand break protein PARP induces differential killing of ATM-null CLL tumours. Here we expand the concept of synthetic lethality in ATM-null CLL and address the question of whether ATM-null deficient CLL cells can be targeted by inhibition of the ATR protein that governs responses to post-replicative damage and co-operates with ATM. First, we addressed the status of the ATR pathway in primary CLL cells and consistent with previous findings we observed that initiation of cell cycling is required for both ATR upregulation and activation of ATR target Chk1 in response to replicating stress inducing agent hydroxyurea. We then proceeded with testing viability of the isogenic CLL cell line CII, with and without stable ATM knock down, in the presence or absence of increasing doses of ATR inhibitor AZD6738. We observed a uniform loss of cellular viability in the presence of 1 or 3 μM of inhibitor in ATM-null cells but not in the ATM-wt counterpart. Similar observation was made in primary CLL cells initiated to cycle in the presence of stimulatory oligonucleotide-ODN2006/IL2 support. To confirm the cytotoxic effect of AZD6738 in vivo we used an ATM null primary CLL xenograft model. Representative primary CLL tumour cells with 15% bialleic ATM inactivation, as assessed by percentage of 11q deletion and allelic frequency of ATM mutation 4220T>C, was engrafted in the presence of activated autologous T lymphocytes into 10 NOG mice. Upon detection of engraftment in peripheral blood, animals were treated by oral administration of either AZD6738 (50mg/kg) or vehicle alone over a 2 week period, and tumour load measured by FACS analysis of CD45+ CD19+ human cells in infiltrated spleens. We observed a reduction in tumour cell numbers in AZD6738-treated compared to vehicle-treated spleens and current investigations are underway to determine whether this difference can be attributed to the selective disappearance of CLL population with biallelic ATM loss. We suggest that targeting ATR pathway provides an attractive approach for selective killing of ATM-null CLL cells and that this approach should be considered as a future therapeutic strategy for this CLL subtype. Disclosures: Off Label Use: ATR inhibitor AZD6738 targets ATM-null phenotype inducing synthetic lethality. Jeff:AstraZeneca Pharmaceuticals: Employment, Patents & Royalties. Lau:AstraZeneca Pharmaceuticals: Employment.

scholarly journals Retention but Not Recruitment of Crb2 at Double-Strand Breaks Requires Rad1 and Rad3 Complexes

2003 ◽  
Vol 23 (17) ◽  
pp. 6150-6158 ◽  
Author(s):  
Li-Lin Du ◽  
Toru M. Nakamura ◽  
Bettina A. Moser ◽  
Paul Russell
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Double Strand Breaks ◽  
Live Cells ◽  
Adapter Protein ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
Checkpoint Signaling ◽  
Strand Breaks ◽  
Checkpoint Protein

ABSTRACT The fission yeast checkpoint protein Crb2, related to budding yeast Rad9 and human 53BP1 and BRCA1, has been suggested to act as an adapter protein facilitating the phosphorylation of specific substrates by Rad3-Rad26 kinase. To further understand its role in checkpoint signaling, we examined its localization in live cells by using fluorescence microscopy. In response to DNA damage, Crb2 localizes to distinct nuclear foci, which represent sites of DNA double-strand breaks (DSBs). Crb2 colocalizes with Rad22 at persistent foci, suggesting that Crb2 is retained at sites of DNA damage during repair. Damage-induced Crb2 foci still form in cells defective in Rad1, Rad3, and Rad17 complexes, but these foci do not persist as long as in wild-type cells. Our results suggest that Crb2 functions at the sites of DNA damage, and its regulated persistent localization at damage sites may be involved in facilitating DNA repair and/or maintaining the checkpoint arrest while DNA repair is under way.

scholarly journals The p400 ATPase regulates nucleosome stability and chromatin ubiquitination during DNA repair

2010 ◽  
Vol 191 (1) ◽  
pp. 31-43 ◽  
Author(s):  
Ye Xu ◽  
Yingli Sun ◽  
Xiaofeng Jiang ◽  
Marina K. Ayrapetov ◽  
Patryk Moskwa ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Double Strand Breaks ◽  
Active Process ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response ◽  
Significant Barrier ◽  
Nucleosome Stability

The complexity of chromatin architecture presents a significant barrier to the ability of the DNA repair machinery to access and repair DNA double-strand breaks (DSBs). Consequently, remodeling of the chromatin landscape adjacent to DSBs is vital for efficient DNA repair. Here, we demonstrate that DNA damage destabilizes nucleosomes within chromatin regions that correspond to the γ-H2AX domains surrounding DSBs. This nucleosome destabilization is an active process requiring the ATPase activity of the p400 SWI/SNF ATPase and histone acetylation by the Tip60 acetyltransferase. p400 is recruited to DSBs by a mechanism that is independent of ATM but requires mdc1. Further, the destabilization of nucleosomes by p400 is required for the RNF8-dependent ubiquitination of chromatin, and for the subsequent recruitment of brca1 and 53BP1 to DSBs. These results identify p400 as a novel DNA damage response protein and demonstrate that p400-mediated alterations in nucleosome and chromatin structure promote both chromatin ubiquitination and the accumulation of brca1 and 53BP1 at sites of DNA damage.

scholarly journals Oocytes can efficiently repair DNA double-strand breaks to restore genetic integrity and protect offspring health

2020 ◽  
Vol 117 (21) ◽  
pp. 11513-11522 ◽  
Author(s):  
Jessica M. Stringer ◽  
Amy Winship ◽  
Nadeen Zerafa ◽  
Matthew Wakefield ◽  
Karla Hutt
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Germ Line ◽  
Genotoxic Stress ◽  
Double Strand Breaks ◽  
Genetic Integrity ◽  
Dna Double Strand Breaks ◽  
Γ Irradiation ◽  
Strand Breaks ◽  
Offspring Health

Female fertility and offspring health are critically dependent on an adequate supply of high-quality oocytes, the majority of which are maintained in the ovaries in a unique state of meiotic prophase arrest. While mechanisms of DNA repair during meiotic recombination are well characterized, the same is not true for prophase-arrested oocytes. Here we show that prophase-arrested oocytes rapidly respond to γ-irradiation–induced DNA double-strand breaks by activating Ataxia Telangiectasia Mutated, phosphorylating histone H2AX, and localizing RAD51 to the sites of DNA damage. Despite mobilizing the DNA repair response, even very low levels of DNA damage result in the apoptosis of prophase-arrested oocytes. However, we show that, when apoptosis is inhibited, severe DNA damage is corrected via homologous recombination repair. The repair is sufficient to support fertility and maintain health and genetic fidelity in offspring. Thus, despite the preferential induction of apoptosis following exogenously induced genotoxic stress, prophase-arrested oocytes are highly capable of functionally efficient DNA repair. These data implicate DNA repair as a key quality control mechanism in the female germ line and a critical determinant of fertility and genetic integrity.

High NaCl causes Mre11 to leave the nucleus, disrupting DNA damage signaling and repair

2003 ◽  
Vol 285 (2) ◽  
pp. F266-F274 ◽  
Author(s):  
Natalia I. Dmitrieva ◽  
Dmitry V. Bulavin ◽  
Maurice B. Burg
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Repair ◽  
Cell Cycle Arrest ◽  
Dna Damage Response ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Cycle Arrest ◽  
Damage Response

High NaCl causes DNA double-strand breaks and cell cycle arrest, but the mechanism of its genotoxicity has been unclear. In this study, we describe a novel mechanism that contributes to this genotoxicity. The Mre11 exonuclease complex is a central component of DNA damage response. This complex assembles at sites of DNA damage, where it processes DNA ends for subsequent activation of repair and initiates cell cycle checkpoints. However, this does not occur with DNA damage caused by high NaCl. Rather, following high NaCl, Mre11 exits from the nucleus, DNA double-strand breaks accumulate in the S and G2 phases of the cell cycle, and DNA repair is inhibited. Furthermore, the exclusion of Mre11 from the nucleus by high NaCl persists following UV or ionizing radiation, also preventing DNA repair in response to those stresses, as evidenced by absence of H2AX phosphorylation at places of DNA damage and by impaired repair of damaged reporter plasmids. Activation of chk1 by phosphorylation on Ser345 generally is required for DNA damage-induced cell cycle arrest. However, chk1 does not become phosphorylated during high NaCl-induced cell cycle arrest. Also, high NaCl prevents ionizing and UV radiation-induced phosphorylation of chk1, but cell cycle arrest still occurs, indicating the existence of alternative mechanisms for the S and G2/M delays. DNA breaks that occur normally during processes such as DNA replication and transcription, as well as damages to DNA induced by genotoxic stresses, ordinarily are rapidly repaired. We propose that inhibition of this repair by high NaCl results in accumulation of DNA damage, accounting for the genotoxicity of high NaCl, and that cell cycle delay induced by high NaCl slows accumulation of DNA damage until the DNA damage-response network can be reactivated.

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