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Results for “Double stranded breaks”

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PubMed1991

Lack of interference of DNA single-strand breaks with the measurement of double-strand breaks in mammalian cells using the neutral filter elution assay.

In this study, the effect of DNA single strand breaks (ssb) on the neutral (pH 9.6) filter elution of DNA from Chinese hamster ovary (CHO K1) cells containing DNA double strand breaks (dsb) was investigated. Protein associated ssb were induced by the inhibition of DNA topoisomerase I with camptothecin (cpt). Protein associated dsb were introduced by treating cells with the DNA topoisomerase II poison; etoposide (VP-16). Protein associated ssb and dsb were converted to ssb and dsb by proteinase K present in the lysis solution. In some experiments dsb were generated by the restriction endonuclease Pvu II. It was found that elution of DNA in the presence and absence of ssb was similar under neutral conditions. This finding is consistent with the view that the fast component of the bi-phasic repair kinetics observed in irradiated mammalian cells with the neutral filter elution technique is not attributable to the interference of ssb with the measurement of dsb, and thus suggests that the two components of repair observed with the neutral filter elution elution technique may represent two different types of dsb or modes of repair of dsb.

Animals↗

PubMed2025

Focus on numbers - characterizing protein accumulation at DNA double-strand breaks.

Unrepaired DNA double-strand breaks can lead to cell death or genomic rearrangements. The DNA damage response (DDR) is a complex signaling cascade in which a plethora of factors act to finely tune repair pathway choice. Several DDR proteins have been shown to accumulate at sites of DNA lesions in characteristic dot-like structures known as DNA repair foci. Changes in foci brightness, commonly expressed in arbitrary intensity units, are often used as readout for DNA repair dynamics. However, due in part to technical challenges, the stoichiometry, absolute number of proteins recruited to DDR foci, and their impact on the resolution of the break remain incompletely characterized. Here, we combine spatial intensity distribution analysis (SpIDA) and a custom foci detection algorithm into an easy-to-use pipeline that, starting from confocal images, allows quantitative description of protein accumulation in DNA repair foci. Moreover, by quantifying foci based on their molecular count, SpIDA overcomes the limitations of ambiguous intensity units, enabling stoichiometric quantification between repair factors and providing a unifying means for experimental comparisons.

DNA Breaks, Double-Stranded↗

PubMed1982

DNA double strand breaks in Ehrlich ascites tumour cells at low doses of x-rays. II. Can cell death be attributed to double strand breaks?

The induction and repair of DNA double strand breaks (dsb) in early stationary Ehrlich ascites tumour cells by X-rays was determined using an improved sedimentation technique in neutral sucrose gradients. The disappearance of dsb was followed during post-irradiation incubation of the cells and was interpreted as dsb repair. Kinetics were approximated by exponential functions with time constants of t37 = 3.0 +/- 0.7 hours ('conditioned' medium) and t37 = 2.0 +/- 0.5 hours (growth medium). Maximal repair was reached after 24 hours and the relationship of the remaining breaks with dose was interpreted on the basis of a recombination repair model. Using these dsb data and on the assumption of one dsb being a lethal event, cell survival curves were calculated for different repair times and compared with experimental curves. It was shown that cell survival curves can be interpreted on the basis of one unrepaired dsb being a lethal event, when dsb repair continues for about 11 hours after plating the cells on nutrient agar.

Animals↗

PubMed1993

Hypertonic treatment does not affect the radiation yield of interphase chromosome breaks in DNA double-strand break repair-deficient xrs-5 cells.

A DNA double-strand break (DSB) repair-deficient CHO cell line, xrs-5, was used to evaluate the effect of hypertonic treatment on the radiation yield of interphase chromosome breaks, as visualized by means of premature chromosome condensation (PCC). For this purpose, interphase chromosome breaks were measured in plateau-phase G1 cells after exposure to 10 Gy X rays using either the standard protocol for PCC induction or a revised protocol that includes a brief (20 min) treatment in hypertonic medium (500 mM NaCl) during chromosome condensation. Experiments at the chromosome level were complemented by experiments at the DNA and the cellular level performed under similar postirradiation conditions. The results obtained were compared to those reported previously for CHO cells. Radiation yields of interphase chromosome breaks, as measured using standard protocols for PCC induction, were higher in xrs-5 than in CHO cells. However, the yields became similar between the two cell lines when a 20-min incubation in hypertonic medium was introduced immediately after fusion with mitotic cells. This equalization was due to the fact that incubation in hypertonic medium did not affect the radiation yield of interphase chromosome breaks in xrs-5 cells but increased it in CHO cells to levels similar to those measured in xrs-5 cells. This result suggests that xrs-5 cells constitutively express as interphase chromosome breaks a subset of DSBs which in repair-proficient CHO cells requires incubation in hypertonic medium for transformation to interphase chromosome breaks. Rejoining of DSBs, when measured in isotonic medium, was incomplete in xrs-5 cells compared to CHO cells. Rejoining of DSBs normally repaired in xrs-5 cells was completely inhibited during treatment in hypertonic medium. Postirradiation treatment in hypertonic medium affected the survival of xrs-5 cells only modestly, suggesting that the sector of PLD (beta-PLD) which required treatment in hypertonic medium for fixation in repair-proficient CHO cells is fixed constitutively in xrs-5 cells. These results are in agreement with a model developed to interpret results obtained in similar experiments with CHO cells (Iliakis et al., Radiat. Res 135, 160-170, 1993). The model postulates that potentiation in CHO cell killing induced by hypertonic treatment is caused by the transformation of a subset of fast-repairing DSBs to irreparable interphase chromosome breaks and assumes that this transformation is equivalent to fixation of beta-PLD.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

3'-Deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin conquers multidrug resistance by rapid influx following higher frequency of formation of DNA single- and double-strand breaks.

The mechanism of action of 3'-deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin (MX2) was examined in a human leukemia cell line (K562) and its Adriamycin (ADM)-resistant subline (K562/ADM). ADM and MX2 showed an equivalent antitumor effect against K562. K562/ADM was highly resistant to ADM. In cellular pharmacokinetic studies, MX2 showed faster and greater influx than did ADM in both K562 and K562/ADM. The efflux of ADM was rapid in K562/ADM but not in K562. On the other hand, the efflux of MX2 was rapid in both cell lines. The formation of DNA single-strand breaks and double-strand breaks by ADM was significantly lower in K562/ADM than K562. On the other hand, formation of those breaks by MX2 was not decreased. Although some of the DNA breaks induced by MX2 were resealed, there was no difference in the degree of resealing in K562 and K562/ADM cells. On the other hand, most of the small number of DNA breaks in K562/ADM induced by ADM were resealed. The topoisomerase II activity in K562 and K562/ADM was not significantly different. It is concluded that MX2 conquers multidrug resistance by rapid influx following a higher frequency of formation of DNA single- and double-strand breaks in K562/ADM cells.

Antibiotics, Antineoplastic↗

PubMed2006

Targeted chromosomal gene modification in human cells by single-stranded oligodeoxynucleotides in the presence of a DNA double-strand break.

A DNA double-strand break (DSB) cannot be tolerated by a cell and is dealt with by several pathways. Here, it was hypothesized that DSB induction close to a targeted mutation in the genome of a mammalian cell might attract oligodeoxynucleotide (ODN)-directed gene repair. A HEK-293-derived cell line had been engineered harboring a single target locus with open reading frames encoding the living-cell reporter proteins LacZ and EGFP, the latter translationally decoupled by a DNA spacer with a unique I-SceI recognition site for defined DSB induction. To enable expression of a fluorescent LacZ-EGFP fusion protein, single-stranded (ss) ODNs (80 or 96 nucleotides long) spanning the DSB were designed to fuse both reading frames by altering a few base-pair positions, deleting 59 bp or introducing a 10-bp fragment. The ssODNs alone generated few EGFP-positive cells. With I-SceI transiently expressed, more than 0.3% of cells revealed EGFP expression 7 days after transfection, with up to 96% of the loci faithfully corrected, depending on the ssODN used. During these correction events, the ssODN did not become physically incorporated into the chromosome, but served only as information template. Unwanted insertional mutagenesis also occurred. Both observations have important implications for gene therapy.

Base Sequence↗

PubMed2004

DNA-dependent protein kinase and XRCC4-DNA ligase IV mobilization in the cell in response to DNA double strand breaks.

Repair of DNA double strand breaks (DSBs) by the non-homologous end joining (NHEJ) pathway in mammals requires at least the DNA-dependent protein kinase (DNA-PK) and the DNA ligase IV-XRCC4 protein complexes. DNA-PK comprises the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs. Here we report the first description of the nuclear mobilization of endogenous NHEJ proteins after exposure of human cells to double strand-breaking agents. DSB infliction specifically induced a dose- and time-dependent mobilization of Ku70/80, DNA-PKcs, XRCC4, and DNA ligase IV proteins from a soluble nucleoplasmic compartment to a less extractable nuclear fraction. XRCC4 recruitment was accompanied by its DNA-PK-dependent phosphorylation. The recruited proteins co-immunoprecipitated, indicating that they had assembled into complexes. However, DNA-PK was attached to chromatin, whereas XRCC4-ligase IV resisted solubilization by DNase I. The rates of appearance and dissolution of NHEJ proteins paralleled that of histone variant H2AX phosphorylation and dephosphorylation. We established that under conditions of genomic DSB infliction 1) Ku recruitment was not dependent on the co-recruitment of the other NHEJ proteins, 2) DNA-PKcs was physically required for the mobilization of the XRCC4-ligase IV complex, 3) DNA ligase IV was physically necessary for stable recruitment of XRCC4, and 4) phosphorylation of either H2AX or XRCC4 was unnecessary for DNA-PK or XRCC4-ligase IV recruitment. Altogether these results offer insights into the interplay between key NHEJ proteins during this repair process in the cell.

Antigens, Nuclear↗

PubMed1992

Pulsed-field gel electrophoresis in the measurement of DNA double-strand break repair in xrs-6 and CHO cell lines: DNA degradation under some conditions interferes with the assessment of double-strand break rejoining.

Pulsed-field gel electrophoresis (PFGE) is a sensitive assay for DNA double-strand breaks (DSBs). The application of PFGE to the measurement of DSB rejoining requires attention to conditions after irradiation. When cells are irradiated and incubated while encapsulated in agarose, DNA DSB rejoining appears incomplete or absent due to DNA degradation artifacts. Using PFGE we have measured the DSB rejoining defect in xrs-6 cells and shown how DNA degradation can affect the results.

Animals↗

PubMed1995

The effect of hydrogen peroxide/L-histidine-induced DNA single- vs. double-strand breaks on poly(ADP-ribose)polymerase.

L-Histidine markedly increases the ability of hydrogen peroxide to induce DNA cleavage and this effect is associated with a 3-aminobenzamide-inhibitable decline in NAD+ levels, an event which very likely reflects an enhanced stimulation of the enzyme poly(ADP-ribose)polymerase. 3-Aminobenzamide slowed down the removal of alkaline elution-detected strand breaks induced by either H2O2 alone (producing only DNA single-strand breaks) or associated with L-histidine (resulting in the formation of both single-strand breaks and DNA double-strand breaks), and the extent of inhibition was similar under the two experimental conditions. 3-Aminobenzamide did not affect the rate of rejoining of DNA double-strand breaks generated by the cocktail H2O2/L-histidine. The above results suggest that these double-strand breaks have hardly any effect on the induction of poly(ADP-ribose)polymerase activity, a conclusion that is consistent with the observation that the activity of this enzyme appears to be basically identical under conditions that abolish the formation of DNA double-strand breaks, in the absence of measurable variations in the level of induction of DNA single-strand breaks (e.g. in the presence of an excess of L-glutamine, a competitive inhibitor of L-histidine uptake). Finally, 3-aminobenzamide did not affect the toxicity of the oxidant, both in the absence and presence of L-histidine.

Animals↗

PubMed2006

NHEJ-deficient DT40 cells have increased levels of immunoglobulin gene conversion: evidence for a double strand break intermediate.

Activation-induced cytidine deaminase (AID) likely initiates immunoglobulin gene-conversion (GC) by deaminating cytidines within the V-region of chicken B-cells. However, the intervening DNA lesion required to initiate GC remains elusive. GC could be initiated by a single strand break or a double strand break (DSB). To distinguish between these possibilities, we examined GC in the chicken DT40 B cell line deficient in non-homologous end joining (NHEJ). It is known that the NHEJ and homologous recombination DNA repair pathways compete for DSBs. In light of this, if a DSB is the major intermediate, deficiency in NHEJ should result in increased levels of GC. Here we show that DNA-PKcs(-/-/-) and Ku70(-/-) DT40 cells had 5- to 10-fold higher levels of GC relative to wildtype DT40 as measured by surface IgM reversion and sequencing of the V-region. These data suggest that a DSB is the major DNA lesion that initiates GC.

Animals↗

PubMed2006

Repair of DNA double strand breaks: in vivo biochemistry.

Double strand breaks (DSBs) can cause damage to the genomic integrity of a cell as well as initiate genetic recombination processes. The HO and I-SceI endonucleases from budding yeast have provided a way to study these events by inducing a unique DSB in vivo under the control of a galactose-inducible promoter. The GAL::HO construct has been used extensively to study processes such as nonhomologous end joining, intra- and interchromosomal gene conversion, single strand annealing and break-induced recombination. Synchronously induced DSBs have also been important in the study of the DNA damage checkpoint, adaptation, and recovery pathways of yeast. This chapter describes methods of using GAL::HO to physically monitor the progression of events following a DSB, specifically the events leading to the switching of mating type by gene conversion of MAT using the silent donors at HML and HMR. Southern blot analysis can be used to follow the overall events in this process such as the formation of the DSB and product. Denaturing alkaline gels and slot blot techniques can be employed to follow the 5' to 3' resection of DNA starting at the DSB. After resection, the 3' tail initiates a homology search and then strand invades its homologous sequence at the donor cassette. Polymerase chain reaction is an important means to assay strand invasion and the priming of new DNA synthesis as well as the completion of gene conversion. Methods such as chromatin immunoprecipitation have provided a means to study many proteins that associate with a DSB, including not only recombination proteins, but also proteins involved in nonhomologous end joining, cell cycle arrest, chromatin remodeling, cohesin function, and mismatch repair.

DNA Damage↗

PubMed1998

Molecular mechanisms of DNA double strand break repair.

DNA double-strand breaks (DSBs) are major threats to the genomic integrity of cells. If not taken care of properly, they can cause chromosome fragmentation, loss and translocation, possibly resulting in carcinogenesis. Upon DSB formation, cell-cycle checkpoints are triggered and multiple DSB repair pathways can be activated. Recent research on the Nijmegen breakage syndrome, which predisposes patients to cancer, suggests a direct link between activation of cell-cycle checkpoints and DSB repair. Furthermore, the biochemical activities of proteins involved in the two major DSB repair pathways, homologous recombination and DNA end-joining, are now beginning to emerge. This review discusses these new findings and their implications for the mechanisms of DSB repair.

Animals↗

PubMed1999

How plants make ends meet: DNA double-strand break repair.

DNA double-strand breaks (DSBs) lead to serious genomic deficiencies if left unrepaired. Recent studies have provided new insight into the mechanisms, the mutants and the genes involved in DSB repair in plants. These studies indicate that high fidelity DSB repair via homologous recombination is less frequent than non-homologous end-joining. Interestingly, non-homologous end-joining in plants is more error-prone than in other species, being associated with various rearrangements that often include deletions and insertions (filler DNA). We discuss the mechanism of error-prone DSB repair, which is probably an important driving force in plant genome evolution.

Journal Article↗

PubMed2003

Regulation and mechanisms of mammalian double-strand break repair.

The double-strand break (DSB) is believed to be one of the most severe types of DNA damage, and if left unrepaired is lethal to the cell. Several different types of repair act on the DSB. The most important in mammalian cells are nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR). NHEJ is the predominant type of DSB repair in mammalian cells, as opposed to lower eucaryotes, but HRR has recently been implicated in critical cell signaling and regulatory functions that are essential for cell viability. Whereas NHEJ repair appears constitutive, HRR is regulated by the cell cycle and inducible signal transduction pathways. More is known about the molecular details of NHEJ than HRR in mammalian cells. This review focuses on the mechanisms and regulation of DSB repair in mammalian cells, the signaling pathways that regulate these processes and the potential crosstalk between NHEJ and HRR, and between repair and other stress-induced pathways with emphasis on the regulatory circuitry associated with the ataxia telangiectasia mutated (ATM) protein.

Animals↗

PubMed2002

Disruption of the Arabidopsis AtKu80 gene demonstrates an essential role for AtKu80 protein in efficient repair of DNA double-strand breaks in vivo.

Double-strand breaks (DSBs) in DNA may occur spontaneously in the cell or be induced experimentally by gamma-irradiation, and represent one of the most serious threats to genomic integrity. Non-homologous end joining (NHEJ) rather than homologous recombination appears to be the major pathway for DSB repair in humans and plants, and it may also be the major route whereby T-DNA integrates into the plant genome during cell transformation. In yeast and mammals, the exposed ends of damaged DNA are bound with high affinity by a dimer of Ku70 and Ku80 proteins, which protects the ends from exonucleases and juxtaposes the two ends of the DSB, independent of sequence homology. Here we report the functional characterization of Ku70 and Ku80 from Arabidopsis thaliana, and demonstrate that AtKu80 and AtKu70 form a heterodimer with DNA binding activity that is specific for DNA ends. An atku80 knockout mutant shows hypersensitivity to the DNA-damaging agents menadione and bleomycin, consistent with a role for AtKu80 in the repair of DSBs in vivo in Arabidopsis.

Amino Acid Sequence↗

PubMed2002

Sensing and repairing DNA double-strand breaks.

The DNA double-strand break (DSB) is the principle cytotoxic lesion for ionizing radiation and radio-mimetic chemicals but can also be caused by mechanical stress on chromosomes or when a replicative DNA polymerase encounters a DNA single-strand break or other type of DNA lesion. DSBs also occur as intermediates in various biological events, such as V(D)J recombination in developing lymphoid cells. Inaccurate repair or lack of repair of a DSB can lead to mutations or to larger-scale genomic instability through the generation of dicentric or acentric chromosomal fragments. Such genome changes may have tumourigenic potential. In other instances, DSBs can be sufficient to induce apoptosis. Because of the threats posed by DSBs, eukaryotic cells have evolved complex and highly conserved systems to rapidly and efficiently detect these lesions, signal their presence and bring about their repair. Here, I provide an overview of these systems, with particular emphasis on the two major pathways of DSB repair: non-homologous end-joining and homologous recombination. Inherited or acquired defects in these pathways may lead to cancer or to other human diseases, and may affect the sensitivity of patients or tumour cells to radiotherapy and certain chemotherapies. An increased knowledge of DSB repair and of other DNA DSB responses may therefore provide opportunities for developing more effective treatments for cancer.

Animals↗

PubMed2005

Tails of histones in DNA double-strand break repair.

DNA double-strand breaks (DSBs) are, arguably, the most deleterious form of DNA damage. An increasing body of evidence points to the inaccurate or inefficient repair of DSBs as a key step in tumorigenesis. Therefore, it is of great importance to understand the processes by which DSBs are detected and repaired. Clearly, these events must take place in the context of chromatin in vivo, and recently, a great deal of progress has been made in understanding the dynamic and active role that histone proteins and chromatin modifying activities play in DNA DSB repair. Here, we briefly review some of the most common techniques in studying DNA DSB responses in vivo, and focus on the contributions of covalent modifications of core histone proteins to these DNA DSB responses.

Amino Acid Sequence↗

PubMed2002

DNA substrate dependence of p53-mediated regulation of double-strand break repair.

DNA double-strand breaks (DSBs) arise spontaneously after the conversion of DNA adducts or single-strand breaks by DNA repair or replication and can be introduced experimentally by expression of specific endonucleases. Correct repair of DSBs is central to the maintenance of genomic integrity in mammalian cells, since errors give rise to translocations, deletions, duplications, and expansions, which accelerate the multistep process of tumor progression. For p53 direct regulatory roles in homologous recombination (HR) and in non-homologous end joining (NHEJ) were postulated. To systematically analyze the involvement of p53 in DSB repair, we generated a fluorescence-based assay system with a series of episomal and chromosomally integrated substrates for I-SceI meganuclease-triggered repair. Our data indicate that human wild-type p53, produced either stably or transiently in a p53-negative background, inhibits HR between substrates for conservative HR (cHR) and for gene deletions. NHEJ via microhomologies flanking the I-SceI cleavage site was also downregulated after p53 expression. Interestingly, the p53-dependent downregulation of homology-directed repair was maximal during cHR between sequences with short homologies. Inhibition was minimal during recombination between substrates that support reporter gene reconstitution by HR and NHEJ. p53 with a hotspot mutation at codon 281, 273, 248, 175, or 143 was severely defective in regulating DSB repair (frequencies elevated up to 26-fold). For the transcriptional transactivation-inactive variant p53(138V) a defect became apparent with short homologies only. These results suggest that p53 plays a role in restraining DNA exchange between imperfectly homologous sequences and thereby in suppressing tumorigenic genome rearrangements.

B-Lymphocytes↗