Detection Of Double-strand Breaks Research Articles

ATR signaling in mammalian meiosis: From upstream scaffolds to downstream signaling

In germ cells undergoing meiosis, the induction of double strand breaks (DSBs) is required for the generation of haploid gametes. Defects in the formation, detection, or recombinational repair of DSBs often result in defective chromosome segregation and aneuploidies. Central to the ability of meiotic cells to properly respond to DSBs are DNA damage response (DDR) pathways mediated by DNA damage sensor kinases. DDR signaling coordinates an extensive network of DDR effectors to induce cell cycle arrest and DNA repair, or trigger apoptosis if the damage is extensive. Despite their importance, the functions of DDR kinases and effector proteins during meiosis remain poorly understood and can often be distinct from their known mitotic roles. A key DDR kinase during meiosis is ataxia telangiectasia and Rad3-related (ATR). ATR mediates key signaling events that control DSB repair, cell cycle progression, and meiotic silencing. These meiotic functions of ATR depend on upstream scaffolds and regulators, including the 9-1-1 complex and TOPBP1, and converge on many downstream effectors such as the checkpoint kinase CHK1. Here, we review the meiotic functions of the 9-1-1/TOPBP1/ATR/CHK1 signaling pathway during mammalian meiosis.

ATM and PRDM9 regulate SPO11-bound recombination intermediates during meiosis

Meiotic recombination is initiated by SPO11-induced double-strand breaks (DSBs). In most mammals, the methyltransferase PRDM9 guides SPO11 targeting, and the ATM kinase controls meiotic DSB numbers. Following MRE11 nuclease removal of SPO11, the DSB is resected and loaded with DMC1 filaments for homolog invasion. Here, we demonstrate the direct detection of meiotic DSBs and resection using END-seq on mouse spermatocytes with low sample input. We find that DMC1 limits both minimum and maximum resection lengths, whereas 53BP1, BRCA1 and EXO1 play surprisingly minimal roles. Through enzymatic modifications to END-seq, we identify a SPO11-bound meiotic recombination intermediate (SPO11-RI) present at all hotspots. We propose that SPO11-RI forms because chromatin-bound PRDM9 asymmetrically blocks MRE11 from releasing SPO11. In Atm–/– spermatocytes, trapped SPO11 cleavage complexes accumulate due to defective MRE11 initiation of resection. Thus, in addition to governing SPO11 breakage, ATM and PRDM9 are critical local regulators of mammalian SPO11 processing.

DNA susceptibility of Saccharomyces cerevisiae to Zeocin depends on the growth phase.

The aim of this study was to evaluate the level of Zeocin-induced double-strand breaks (DSBs) in Saccharomyces cerevisiae cells in a different growth phase, using constant-field gel electrophoresis (CFGE). Saccharomyces cerevisiae diploid strain D7ts1 with enhanced cellular permeability was used. The effects of growth phase and treatment time were evaluated based on Zeocin-induced DSBs, measured by CFGE. Survival assay was also applied. No protoplast isolation was necessary for the detection of DSBs in strain D7ts1. Differences in the response of cells depending on the growth phase were obtained. Cells in exponential growth phase had increased DSB levels only after Zeocin treatment with concentrations equal or higher than 200μgml-1. Increasing treatment time did not result in higher DSB levels. Oppositely, treatment of cells at the beginning of stationary phase with Zeocin concentrations resulted in more than 1.5-fold increase in DSB levels in comparison with those in untreated cells. Increased DSB levels were measured for all the treatment times. A dose-dependent decrease in cell survival was observed after Zeocin treatment with concentrations in the range of lethality LD20-LD50. A strong negative correlation was calculated between the levels of DSBs and cell survival. New information is provided concerning DNA susceptibility depending on the growth phase. DNA susceptibility is higher in cells at the beginning of stationary phase than those in exponential phase. Data presented here illustrate that the optimized by us CFGE protocol is sensitive and could be used successfully for DSB measurement in Saccharomyces cerevisiae strains with enhanced cellular permeability.

The possibilities of pesticide genotoxicity assessment in bovine lymphocytes cultures

The exposure of dairy cattle to genomic substances may result in mutations, metabolic disorders, immunosuppression and reduced fertility. Potential pesticide genotoxicity can be tested in cultured bovine lymphocyte by broad spectrum of methods including chromosomal aberrations (CA), staining bovine chromosomes with fluorescently labelled DNA probes, micronucleus test (MN test), SCE (Sister Chromatid Exchanges), Comet assay, real-time PCR for detection of changes in expression of some genes involved in xenobiotic metabolism, detection of DSB (double-stranded breaks) and biophysical methods of interaction of pesticide compounds with DNA. Keywords: cattle, lymphocytes, pesticides, genotoxicity assessment methods The study was supported by the Slovak Scientific Agency VEGA projects 1/0043/15 and 1/0176/16

TRAX mediates the protective role of the A2A adenosine receptor in detecting the DNA damage induced by oxidative stress

The translin‐associated protein X (TRAX) was originally identified as an interacting protein of Translin, a DNA/RNA binding protein that controls mRNA transport, translation, DNA repair, and DNA recombination. It interacts specifically with an activator (i.e., C1D) of DNA‐dependent protein kinase (DNA‐PK) upon γ‐irradiation and thus is implicated in repair of double‐strand breaks (DSBs). We recently reported that TRAX interacts with phosphorylated ATM (Ser1981) and contributes to the ATM‐mediated DNA repair in response to genotoxic stresses. The A2A adenosine receptor (A2AR) is a G‐protein‐coupled receptor that binds to adenosine and exists in many brain areas. Using a yeast two‐hybrid approach, we found that TRAX is an interacting protein of the cytoplasmic C terminus of A2AR, and mediates the neuritogenesis process via KIF2A. Given that stimulation of A2AR by two A2AR‐selective agonists markedly ameliorates the DSBs evoked by elevated oxidative stress in human iPSCs‐derived neurons, the role of TRAX in mediating the protective role of A2AR is of great interest. In the present study, we present evidence to suggest that TRAX is required for the A2AR‐mediated facilitation of DNA repair in a neuronal cell line (PC12) and primary neurons. Stimulation of A2AR appears to enhance the non‐homologous end joining (NHEJ) repair activity by increasing the activation and phosphorylation of DNA‐PK at Thr2609. Collectively, TRAX contributes to the activation of DNA‐PK and the detection of DSBs evoked by oxidative stress.

Eukaryotic DNA damage checkpoint activation in response to double-strand breaks

Double-strand breaks (DSBs) are the most detrimental form of DNA damage. Failure to repair these cytotoxic lesions can result in genome rearrangements conducive to the development of many diseases, including cancer. The DNA damage response (DDR) ensures the rapid detection and repair of DSBs in order to maintain genome integrity. Central to the DDR are the DNA damage checkpoints. When activated by DNA damage, these sophisticated surveillance mechanisms induce transient cell cycle arrests, allowing sufficient time for DNA repair. Since the term "checkpoint" was coined over 20 years ago, our understanding of the molecular mechanisms governing the DNA damage checkpoint has advanced significantly. These pathways are highly conserved from yeast to humans. Thus, significant findings in yeast may be extrapolated to vertebrates, greatly facilitating the molecular dissection of these complex regulatory networks. This review focuses on the cellular response to DSBs in Saccharomyces cerevisiae, providing a comprehensive overview of how these signalling pathways function to orchestrate the cellular response to DNA damage and preserve genome stability in eukaryotic cells.

Validation and application of Drosophila melanogaster as an in vivo model for the detection of double strand breaks by neutral Comet assay

Comet assay under neutral conditions allows detection of DNA double-strand breaks (DSBs), which has consequence to genome instability and carcinogenesis. The present study aims to validate the neutral Comet assay for genotoxicity assessment in Drosophila melanogaster (Oregon R +) with three well known DSBs inducers i.e. cyclophosphamide (CP), bleomycin (BLM), cisplatin (CPT) and subsequently its efficacy in detecting DSBs in the organism exposed to a well known environmental chemical, chromium [Cr(VI)]. Third instar larvae of D. melanogaster were fed different concentrations of BLM, CPT and CP (50.0–200.0 μg/ml) or Cr(VI) (5.0–20.0 μg/ml) mixed standard Drosophila food for 48 h. Neutral Comet assay was performed in cells of mid gut and brain from control and treated larvae. Our results show a dose-dependent increase in the migration of DNA in cells of the exposed organisms. A comparison among DNA lesions per mole number of the test chemical in the exposed groups showed that both BLM and CPT induce more DSBs than CP. Interestingly, Cr(VI) at 20.0 μg/ml was found to induce significantly increased ( p < 0.001) DSBs in the exposed organism as compared to the control. The study while validating neutral Comet assay in D. melanogaster suggests its use for in vivo assessment of environmental chemical induced DSBs.

Cellular responses to targeted genomic sequence modification using single-stranded oligonucleotides and zinc-finger nucleases

Single-stranded oligonucleotides (ssODNs) and zinc-finger nucleases (ZFNs) are two approaches that are being pursued to achieve sequence specific genome modification. ZFNs induce high rates of homologous recombination (HR) between the target sequence and a given donor by introducing site-specific genomic double-strand breaks (DSBs). The mode of action that is used by ssODNs remains largely unknown, but may involve genomic integration of the ssODNs. In this work, cellular responses following ssODN and ZFN mediated correction of a genomic reporter gene have been investigated in human cells. Comparison of the cell cycle distribution of corrected cells following ssODN or ZFN exposure, established that ssODN corrected cells were arrested in the late S and G2/M cell cycle phases, while ZFN corrected cells displayed normal cell cycle profiles. We demonstrate that after ssODN mediated gene correction, phosphorylation of the damage sensor protein H2AX could be observed in 5.8% and 29% of the corrected cells, using a single copy and a multi copy reporter, respectively. When using the ZFN strategy in a single copy reporter only 1.5% of the corrected cells were positive for γ-H2AX staining. By direct detection of genomic DSBs we establish that the observed cell cycle arrest following ssODN mediated gene correction could be associated with the presence of unrepaired genomic DSBs. Lastly, we establish that although a mutant cellular mismatch repair (MMR) system as expected enhanced ssODN mediated gene correction, the capacity of the ssODN corrected cells to proliferate was not influenced by the MMR system. In conclusion gene correction by means of the ssODN strategy leads to activation of DNA damage signalling and cell cycle arrest due to formation of unrepaired genomic DSBs in a high proportion of the corrected cells. On the contrary, cells corrected using ZFNs displayed normal cell cycle distribution and lower rates of DNA damage.

Generation of phosphorylated histone H2AX by benzene metabolites

Benzene is a well known environmental carcinogen which causes myeloid leukemia. DNA damage induced by benzene metabolites such as hydroquinone (HQ) and p-benzoquinone (BQ) is one reason for the leukemogenesis. In this study, we showed that treatment with HQ and BQ quickly and clearly generated phosphorylated histone H2AX (gamma-H2AX) which has been recently considered an index of the production of double strand breaks (DSBs). HQ and BQ produced discrete foci of gamma-H2AX within the nucleus of HL-60 cells in a dose-dependent manner. gamma-H2AX appeared after the treatment with HQ and BQ for 2h, and increased time-dependently up to 4-8h. HQ and BQ increased intracellular oxidation, and an antioxidant, N-acetylcysteine, clearly inhibited the phosphorylation, suggesting that reactive oxygen species produced from HQ and BQ contributed to the generation. gamma-H2AX was sensitively detected after treatment with low concentrations of HQ and BQ, compared with the direct detection of DSBs by biased sinusoidal field gel electrophoresis and with the assessment of cytotoxicity based on cell survival. DSBs are the most serious form of DNA damage and are associated with genomic instability leading to myeloid leukemia. gamma-H2AX may be a useful tool for judging the genotoxicity of benzene metabolites sensitively.

Processing of clustered DNA damage in human breast cancer cells MCF-7 with partial DNA-PKcs deficiency

Complex DNA damage such as double strand breaks (DSBs) and non-DSB bistranded oxidative clustered DNA lesions (OCDL) (two or more DNA lesions within a short DNA fragment of 1–10 bp on opposing DNA strands) are considered the hallmark of ionizing radiation. Clustered DNA lesions are hypothesized to be repair-resistant lesions challenging the repair mechanisms of the cell. The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays an important role during the processing of DSBs. To evaluate the role of DNA-PKcs in the processing of complex DNA damage in human MCF-7 breast cancer cells we used small interfering RNAs (siRNAs) to target the silencing of the gene Prkdc coding for DNA-PKcs. MCF-7 cells with knockdown DNA-PKcs expression showed a marked decrease in their efficiency to process DSBs and OCDL after exposure to radiotherapy-relevant γ ray doses. For the detection and measurement of complex DSBs and OCDL, we used the γ-H2AX assay and an adaptation of pulsed field gel electrophoresis with Escherichia coli repair enzymes as DNA damage probes. An accumulation of all types of DNA damage was detected for the siRNA-treated MCF-7 cells compared to controls. These findings point to the important role of DNA-PKcs in the processing of complex DNA damage and its potential association with breast cancer development.

46. The Combination of Oncolytic Adenoviruses and Ionizing Radiation Inhibits DNA Double Strand Break Detection Which Results in Autophagocytotic Prostate Cancer Cell Death

46. The Combination of Oncolytic Adenoviruses and Ionizing Radiation Inhibits DNA Double Strand Break Detection Which Results in Autophagocytotic Prostate Cancer Cell Death

DNA Damage Recognition Proteins Localize along Heavy Ion Induced Tracks in the Cell Nucleus

To identify the repair dynamics involved in high linear energy transfer (LET) radiation-induced DNA damage, phospho-H2AX (gammaH2AX) foci formation was analyzed after cellular exposure to iron ions (Fe-ions, 500 MeV u(-1), 200 KeV microm(-1)). The foci located at DNA damage sites were visualized using immunocytochemical methods. Since H2AX is phosphorylated at sites of radiation-induced double strand breaks (DSB), gammaH2AX foci were used to detect or illuminate tracks formed by DSB after exposure to various doses of ionizing radiation. Additional DSB-recognition proteins such as ATM phospho-serine 1981, DNA-PKcs phospho-threonine 2609, NBS1 phospho-serine 343 and CHK2 phospho-threonine 68 all co-localized with gammaH2AX at high LET radiation induced DSB. In addition, Fe-ion induced foci remained for longer times than X-radiation induced foci. These findings suggest that Fe-ion induced damage is repaired more slowly than X-radiation induced damage, possibly because Fe-ion induced damage or lesions are more complex or extensive. Antibodies for all these phosphorylated DNA DSB recognition proteins appear to be very effective for the detection and localization of DSB.

Detection of Complex DNA Damage in γ-Irradiated Acute Lymphoblastic Leukemia Pre-B NALM-6 Cells

Bistranded complex DNA damage, i.e., double-strand breaks (DSBs) and non-DSB oxidative clustered DNA lesions, is hypothesized to challenge the repair mechanisms of the cell and consequently the genomic integrity. The oxidative clustered DNA lesions may be persistent and may accumulate in human cancer cells for long times after irradiation. To evaluate the detection and possible accumulation of oxidative clustered DNA lesions in leukemia cells exposed to doses equivalent to those used in radiotherapy, we measured the induction of DSBs and three different types of oxidative clustered DNA lesions in NALM-6 cells, a human acute lymphoblastic leukemia (ALL) pre-B cell line, after exposure to (137)Cs gamma rays. For the detection and measurement of DSBs and oxidative clustered DNA lesions, we used an adaptation of the neutral comet assay (single-cell gel electrophoresis) using E. coli repair enzymes (Endo IV, Fpg and Endo III) as enzymatic probes. We found a linear dose response for the induction of DSBs and oxidative clustered DNA lesions. Clustered DNA lesions were more prevalent than prompt DSBs. For each DSB induced by radiation, approximately 2.5 oxidative clustered DNA lesions were detected. To our knowledge, this is the first study to demonstrate the detection and linear induction of oxidative clustered DNA lesions with radiation dose in an ALL cell line. These results point to the biological significance of clustered DNA lesions.

Induction and processing of complex DNA damage in human breast cancer cells MCF-7 and nonmalignant MCF-10A cells

Oxidatively induced stress and DNA damage have been associated with various human pathophysiological conditions, including cancer and aging. Complex DNA damage such as double-strand breaks (DSBs) and non-DSB bistranded oxidatively induced clustered DNA lesions (OCDL) (two or more DNA lesions within a short DNA fragment of 1–10 bp on opposing DNA strands) are hypothesized to be repair-resistant lesions challenging the repair mechanisms of the cell. To evaluate the induction and processing of complex DNA damage in breast cancer cells exposed to radiotherapy-relevant γ-ray doses, we measured single-strand breaks (SSBs), DSBs, and OCDL in MCF-7 and HCC1937 malignant cells as well as MCF-10A nonmalignant human breast cells. For the detection and measurement of SSBs, DSBs, and OCDL, we used the alkaline single-cell gel electrophoresis, γ-H2AX assay, and an adaptation of pulsed-field gel electrophoresis with E. coli repair enzymes as DNA damage probes. Increased levels for most types of DNA damage were detected in MCF-7 cells while the processing of DSBs and OCDL was deficient in these cells compared to MCF-10A cells. Furthermore, the total antioxidant capacity of MCF-7 cells was lower compared to their nonmalignant counterparts. These findings point to the important role of complex DNA damage in breast cancer and its potential association with breast cancer development especially in the case of deficient BRCA1 expression.

The 12/23 rule is enforced at the cleavage step of V(D)J recombination in vivo.

V(D)J recombination is initiated by the introduction of double-stranded breaks (DSB) adjacent to recombination signal sequences (RSS). Each RSS contains a conserved heptamer and a conserved nonamer element separated by a 12 or 23 nucleotide spacer. In vivo, efficient recombination requires one RSS of each spacer length, although it has been unclear whether this '12/23 rule' regulates cleavage, joining, or both. We describe a novel system that permits semiquantitative detection of DSB at RSS derived from V(D)J recombination substrates transfected into cultured cells. This approach provides a powerful new tool for analysis of the cleavage and joining steps of V(D)J recombination in vivo. In this study, substrates containing either a consensus 12/23 RSS pair or various deviations from the consensus were used to investigate the requirements for cleavage. The results show that both a 12-spacer and a 23-spacer RSS are required for efficient cleavage. Truncated RAG-1 and RAG-2 proteins, while capable of cleaving at isolated RSS in cell-free systems, also require a 12/23 RSS pair for efficient cleavage in vivo. These results suggest that the 12/23 rule is enforced at or prior to cleavage and support a synapsis model for V(D)J recombination. Detection of rare cleavage events in substrates containing a single RSS or a pair of RSS with the same spacer length provide evidence for an inefficient, single RSS cleavage pathway that may contribute to aberrant V(D)J rearrangements in vivo.