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Results for “Double stranded breaks”

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PubMed1994

Visualization of DNA double strand breaks in the gerbil hippocampal CA1 following transient ischemia.

To clarify the relationship between delayed neuronal death and apoptosis, we made an attempt to detect DNA strand breaks in the gerbil hippocampal CA1 pyramidal neurons following transient ischemia by an in situ DNA double strand breaks staining. The condensed nuclei of neurons in CA1 were occasionally seen and positively stained 3 days after ischemia. The degenerating dark neurons with condensed nuclei in CA1 markedly increased in number and were clearly stained for DNA strand breaks 5 days after ischemia, whereas normal-looking nuclei remained unstained. No neurons were stained in other hippocampal subfields and dentate gyrus. The present study strongly indicates that the CA1 pyramidal neurons die through an apoptotic process.

Animals↗

PubMed2006

ATM-mediated response to DNA double strand breaks in human neurons derived from stem cells.

Ataxia-telangiectasia (A-T) is a multi-system genomic instability syndrome that is caused by loss or inactivation of the ATM protein kinase. ATM is largely nuclear in proliferating cells, and activates an extensive network of pathways in response to double strand breaks (DSBs) in the DNA by phosphorylating key proteins in these pathways. The prominent symptom of A-T is neuronal degeneration, making the elucidation of ATM's functions in neurons essential to understanding the disease. It has been suggested that ATM is cytoplasmic in neurons and functions in processes that are not associated with the DNA damage response. Recently we showed that in human neuron-like cells obtained by in vitro differentiation of neuroblastomas, ATM was largely nuclear and mediated the DSB response as in proliferating cells. We have now extended these studies to two additional model systems: neurons derived from human embryonic stem cells, and cortical neurons derived from neural stem cells. The results substantiate the notion that ATM is nuclear in human neurons and mediates the DSB response, the same as it does in proliferating cells. We present here unique and powerful model systems to further study the ATM-mediated network in neurons.

Ataxia Telangiectasia↗

PubMed1996

Double-strand break repair in the absence of RAD51 in yeast: a possible role for break-induced DNA replication.

In wild-type diploid cells of Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break (DSB) at the MAT locus can be efficiently repaired by gene conversion using the homologous chromosome sequences. Repair of the broken chromosome was nearly eliminated in rad52delta diploids; 99% lost the broken chromosome. However, in rad51delta diploids, the broken chromosomes were repaired approximately 35% of the time. None of these repair events were simple gene conversions or gene conversions with an associated crossover, instead, they created diploids homozygous for the MAT locus and all markers in the 100-kb region distal to the site of the DSB. In rad51delta diploids, the broken chromosome can apparently be inherited for several generations, as many of these repair events are found as sectored colonies, with one part being repaired and the other part being lost the broken chromosome. Similar events occur in about 2% of wild-type cells. We propose that a broken chromosome end can invade a homologous template in the absence of RAD51 and initiate DNA replication that may extend to the telomere, 100 or more kb away. Such break-induced replication appears to be similar to recombination-initiated replication in bacteria.

Chromosomes, Fungal↗

PubMed2006

Conservative repair of a chromosomal double-strand break by single-strand DNA through two steps of annealing.

The repair of chromosomal double-strand breaks (DSBs) is essential to normal cell growth, and homologous recombination is a universal process for DSB repair. We explored DSB repair mechanisms in the yeast Saccharomyces cerevisiae using single-strand oligonucleotides with homology to both sides of a DSB. Oligonucleotide-directed repair occurred exclusively via Rad52- and Rad59-mediated single-strand annealing (SSA). Even the SSA domain of human Rad52 provided partial complementation for a null rad52 mutation. The repair did not involve Rad51-driven strand invasion, and moreover the suppression of strand invasion increased repair with oligonucleotides. A DSB was shown to activate targeting by oligonucleotides homologous to only one side of the break at large distances (at least 20 kb) from the break in a strand-biased manner, suggesting extensive 5' to 3' resection, followed by the restoration of resected DNA to the double-strand state. We conclude that long resected chromosomal DSB ends are repaired by a single-strand DNA oligonucleotide through two rounds of annealing. The repair by single-strand DNA can be conservative and may allow for accurate restoration of chromosomal DNAs with closely spaced DSBs.

Chromosomes, Fungal↗

PubMedDate not supplied

Homologous recombination-mediated double-strand break repair in mouse testicular extracts and comparison with different germ cell stages.

Homologous recombination (HR) is established as a significant contributor to double-strand break (DSB) repair in mammalian somatic cells; however, its role in mammalian germ cells has not been characterized, although being conservative in nature it is anticipated to be the major pathway in germ cells. The germ cell system has inherent limitations by which intact cell approaches are not feasible. The present study, therefore, investigates HR-mediated DSB repair in mouse germ cell extracts by using an in vitro plasmid recombination assay based on functional rescue of a neomycin (neo) gene. A significantly high-fold increase in neo+ (Kan(R)) colonies following incubation of two plasmid substrates (neo delta1 and neo delta2) with testicular extracts demonstrated the extracts' ability to catalyze intermolecular recombination. A significant enhancement in recombinants upon linearization of one of the plasmids suggested the existence of an HR-mediated DSB repair activity. Comparison of the activity at sequential developmental stages, spermatogonia, spermatocytes and spermatids revealed its presence at all the stages; spermatocyte being the most proficient stage. Further, restriction analysis of recombinant plasmids indicated the predominance of gene conversion in enriched spermatocytes (mostly pachytenes), in contrast to gonial and spermatid extracts that showed higher reciprocal exchange. In conclusion, this study demonstrates HR repair activity at all stages of male germ cells, suggesting an important role of HR-mediated DSB repair during mammalian spermatogenesis. Further, the observed preference of gene conversion over reciprocal exchange at spermatocyte stage correlates with the close association of gene conversion with the meiotic recombination program.

Animals↗

PubMed1998

A rapid method to monitor repair and mis-repair of DNA double-strand breaks by using cell extracts of the yeast Saccharomyces cerevisiae.

We present a rapid in vitro method to scan the repair of DNA double-strand breaks (DSBs). A DSB was introduced at the EcoRI site within the lacZ gene of the plasmid pUC18 and the plasmid was exposed to cellular extracts from a wild-type repair-competent (RAD) and a mutant (rad52Delta) strain of the yeast Saccharomyces cerevisiae. The fidelity of rejoining was determined by the expression of the lacZ gene after bacterial transformation with the treated plasmid. A cellular extract from the yeast S. cerevisiae was found to be capable of rejoining DNA DSBs. Breaks at the EcoRI site were rejoined by extracts from both wild-type and mutant strains to form circular plasmids with almost equal efficiency. However, the fidelity of rejoining was lower for the rad52Delta extract than for normal wild-type.

DNA↗

PubMed2005

Does gammaH2AX foci formation depend on the presence of DNA double strand breaks?

H2AX is a histone variant that is systematically found and ubiquitously distributed throughout the genome. Since it has been reported that DNA double-strand breaks (DSBs) induce phosphorylation of H2AX at serine 139 (gammaH2AX), an immunocytochemical assay with antibodies recognizing gammaH2AX has become the gold standard for the detection of DSBs. This assay is quite sensitive and is a specific indicator for the existence of a DSB. Until now, it has been reported that various kinds of physical, chemical, and biological factors induce the formation of the gammaH2AX foci detected using this assay. Even when gammaH2AX foci were detected, it was not always possible to conclude that the detected DSBs were produced by environmental stresses in the absence of any known radiation. In this review, emphasis is on discussing whether gammaH2AX foci formation depends on the formation of DSBs.

Animals↗

PubMed2006

Analysis of DNA double-strand break repair pathways in mice.

During the last years significant new insights have been gained into the mechanism and biological relevance of DNA double-strand break (DSB) repair in relation to genome stability. DSBs are a highly toxic DNA lesion, because they can lead to chromosome fragmentation, loss and translocations, eventually resulting in cancer. DSBs can be induced by cellular processes such as V(D)J recombination or DNA replication. They can also be introduced by exogenous agents DNA damaging agents such as ionizing radiation or mitomycin C. During evolution several pathways have evolved for the repair of these DSBs. The most important DSB repair mechanisms in mammalian cells are nonhomologous end-joining and homologous recombination. By using an undamaged repair template, homologous recombination ensures accurate DSB repair, whereas the untemplated nonhomologous end-joining pathway does not. Although both pathways are active in mammals, the relative contribution of the two repair pathways to genome stability differs in the different cell types. Given the potential differences in repair fidelity, it is of interest to determine the relative contribution of homologous recombination and nonhomologous end-joining to DSB repair. In this review, we focus on the biological relevance of DSB repair in mammalian cells and the potential overlap between nonhomologous end-joining and homologous recombination in different tissues.

Animals↗

PubMed2000

DNA ends: maintenance of chromosome termini versus repair of double strand breaks.

This review focuses on the factors that define the differences between the two types of DNA ends encountered by eukaryotic cells: telomeres and double strand breaks (DSBs). Although these two types of DNA termini are functionally distinct, recent studies have shown that a number of proteins is shared at telomeres and sites of DSB repair. The significance of these common components is discussed, as well as the types of DNA repair events that can compensate for a defective telomere.

Chromosomes↗

PubMed1998

Differential inhibitory effect of OK-1035 on DNA repair in L5178Y murine lymphoma sublines with functional or defective repair of double strand breaks.

Radiosensitive L5178Y-S (LY-S) subline and its parental, more radioresistant L5178Y-R (LY-R) subline differ in DNA double strand break (DSB) rejoining. In this work we examined by comet assay the repair of X-ray-induced DNA damage in LY cells treated with OK-1035, a potent DNA-PK inhibitor. The unirradiated cells differ: the respective tail moment values for LY-R and LY-S cells were 9.62+/-2.84 and 3.52+/-0.1, reflecting the susceptibility to lysis conditions as well as the possible endogenous (oxidative) damage level. The level of initial DNA damage measured after irradiation (8 Gy) at DNA-denaturing pH was the same in both LY sublines: the mean tail moment values +/- SD were 92.93+/-10.39 for LY-R cells and 94.93+/-12.94 for LY-S cells. In LY-S cells the repair of 8 Gy X-ray-induced damage proceeded identically in the presence or absence of 2 mM OK-1035 to the same level of residual damage. In contrast, the level of residual damage in inhibitor treated LY-R cells was considerably higher than that in the untreated cells. Moreover, the inhibitor affected LY-R cells in G1 and S phases and not those in G2, in agreement with cell-cycle specificity of DNA-PK. These results may indicate that the DSB repair defect previously identified in LY-S cells is due to a lack of function of DNA-PK or its impaired activation in the irradiated cells.

Animals↗

PubMed1997

Mammalian DNA double-strand break repair protein XRCC4 interacts with DNA ligase IV.

BACKGROUND: Mammalian cells deficient in the XRCC4 DNA repair protein are impaired in DNA double-strand break repair and are consequently hypersensitive to ionising radiation. These cells are also defective in site-specific V(D)J recombination, a process that generates the diversity of antigen receptor genes in the developing immune system. These features are shared by cells lacking components of the DNA-dependent protein kinase (DNA-PK). Although the XRCC4 gene has been cloned, the function(s) of XRCC4 in DNA end-joining has remained elusive. RESULTS: We found that XRCC4 is a nuclear phosphoprotein and was an effective substrate in vitro for DNA-PK. Human XRCC4 associated extremely tightly with another protein(s) even in the presence of 1 M NaCl. Co-immunoprecipitation and adenylylation assays demonstrated that this associated factor was the recently identified human DNA ligase IV. Consistent with this, XRCC4 and DNA ligase IV copurified exclusively and virtually quantitatively over a variety of chromatographic steps. Protein mapping studies revealed that XRCC4 interacted with ligase IV via the unique carboxy-terminal ligase IV extension that comprises two tandem BRCT (BRCA1 carboxyl terminus) homology motifs, which are also found in other DNA repair-associated factors and in the breast cancer susceptibility protein BRCA1. CONCLUSIONS: Our findings provide a function for the carboxy-terminal region of ligase IV and suggest that BRCT domains of other proteins may mediate contacts between DNA repair components. In addition, our data implicate mammalian ligase IV in V(D)J recombination and the repair of radiation-induced DNA damage, and provide a model for the potentiation of these processes by XRCC4.

Animals↗

PubMed1995

Covalent protein-DNA complexes at the 5' strand termini of meiosis-specific double-strand breaks in yeast.

During meiosis in Saccharomyces cerevisiae, the first chemical step in homologous recombination is the occurrence of site-specific DNA double-strand breaks (DSBs). In wild-type cells, these breaks undergo resection of their 5' strand termini to yield molecules with 3' single-stranded tails. We have further characterized the breaks that accumulate in rad50S mutant stains defective in DSB resection. We find that these DSBs are tightly associated with protein via what appears to be a covalent linkage. When genomic DNA is prepared from meiotic rad50S cultures without protease treatment steps, the restriction fragments diagnostic of DSBs selectively partition to the organic-aqueous interphase in phenol extractions and band at lower than normal density in CsCl density gradients. Selective partitioning and decreased buoyant density are abolished if the DNA is treated with proteinase K prior to analysis. Similar results are obtained with sae2-1 mutant strains, which have phenotypes identical to rad50S mutants. The protein is bound specifically to the 5' strand termini of DSBs and is present at both 5' ends in at least a fraction of breaks. The stability of the complex to various protein denaturants and the strand specificity of the attachment are most consistent with a covalent linkage to DSB termini. We propose that the DSB-associated protein is the catalytic subunit of the meiotic recombination initiation nuclease and that it cleaves DNA via a covalent protein-DNA intermediate.

DNA, Fungal↗

PubMed2000

The role of higher-order chromatin structure in the yield and distribution of DNA double-strand breaks in cells irradiated with X-rays or alpha-particles.

PURPOSE: To determine whether the non-random distributions of DNA double-strand breaks in cells observed after alpha-particle irradiation are related to the higher-order structure of the chromatin within the nucleus. MATERIALS AND METHODS: Chinese hamster V79 cells were irradiated as either cellular monolayers, nuclear monolayers with condensed chromatin or nuclear monolayers with relaxed chromatin, and the yields and distribution of DSB measured using two pulsed-field gel electrophoresis protocols capable of separating fragments of 10kbp to 5.7 Mbp. RESULTS: Using conventional FAR analysis, the effect of isolating nuclear monolayers and changing the chromatin condensation state was less for alpha-particle irradiated substrates than for X-irradiated ones. When the total number of breaks was measured by separating and quantifying all the fragments produced in the 10kbp to 5.7Mbp region, the difference between the observed yields of breaks in X-irradiated cells (7.3 x 10(-9) DSB/Gy/bp) relative to alpha-particles (12.1 x 10(-9) DSB/Gy/bp) was largely removed when nuclear monolayers with decondensed chromatin were exposed. The yields, although similar, increased to 44.4 x 10(-9) DSB/Gy/bp for X-irradiated decondensed nuclear monolayers and 46.6 x 10(-9) DSB/Gy/bp for alpha-particle irradiated monolayers. However, the alpha-particle DSB distributions remained non-random. CONCLUSIONS: Our results suggest that the non-random distribution of breaks observed in cells with alpha-particle irradiation, which leads to a high probability for the production of regionally multiply damaged sites, is not related to the underlying chromatin condensation state present in the nucleus.

Alpha Particles↗

PubMed1991

Comparison between pulsed-field and constant-field gel electrophoresis for measurement of DNA double-strand breaks in irradiated Chinese hamster ovary cells.

Pulsed-field gel electrophoresis (PFGE) is one of the most sensitive methods for detecting DNA double-strand breaks in mammalian cells. However, it has been observed that constant-field gel electrophoresis (CFGE), when optimized, can detect breaks with equal efficiency. The migration of DNA from the well and the separation of DNA molecules according to size appear to be different processes; only the latter requires the application of PFGE. CFGE is very sensitive and can detect DNA damage produced by less than 5 Gy of radiation. Low voltage (ca. 0.6 V/cm) during electrophoresis appears to be essential for the migration of the largest fraction of DNA from the agarose plug containing the cells; the electrophoresis run time, cell density in the plug, agarose concentration, nature of detergent and extent of radiolabelling are less important. It is concluded that CFGE is equally sensitive but more rapid and economical than PFGE for the measurement of DNA damage.

Animals↗

PubMed1992

Effect of 2-deoxy-D-glucose on DNA double strand break repair, cell survival and energy metabolism in euoxic Ehrlich ascites tumour cells.

Effects of 2-deoxy-D-glucose (2-DG) on DNA double strand break (dsb) repair, cell survival and on the energy metabolism were investigated in exponentially growing Ehrlich ascites tumour (EAT) cells. Cells in suspension were exposed to 40 Gy of X-rays and allowed to repair (up to 4 h) with or without 2-DG at 37 degrees C. DNA dsb rejoining was measured by means of clamped homogeneous electric field (CHEF), a pulsed field gel electrophoresis technique. The fraction of activity released (FAR) during electrophoresis (DNA associated 14C-thymidine) was used as a parameter to determine the number of dsb present in the DNA. Biphasic kinetics for dsb repair were observed. The presence of 2-DG significantly inhibited the slow component of dsb repair. The presence of 2-DG also enhanced radiation-induced cell killing. ATP content of cells was measured by a bioluminescence method. ATP content in exponentially growing cells was about 4 pg per cell. The level of ATP was reduced by 50% in presence of 2-DG (C2-DG/CG = 1.0).

Adenosine Triphosphate↗

PubMed1994

Simple and complex double-strand breaks induced by electrons.

Biophysical modelling of DNA damage based on Monte Carlo simulation of charged particle tracks allows to describe radiation induced double-strand breaks (dsb) in a quantitative and qualitative way. Experimental and calculated data suggest that in the electron energy range from 50 eV to 1 MeV dsb can be grouped in simple and complex dsb. Complex dsb are mainly produced by low energy electrons with initial energies between approximately 200 and approximately 500 eV, whereas simple dsb are preferentially induced by energy transfers < 200 eV, which produce at least two ionizations.

DNA Damage↗

PubMed1996

Protective effect of ginseng on radiation-induced DNA double strand breaks and repair in murine lymphocytes.

We have examined the effects of ginseng on the induction and repair of gamma-ray-induced DNA double strand breaks (dsb) using neutral filter elution technique at pH 9.6 in cultured murine spleen lymphocytes. Ginseng water extract 500 micrograms/ml was added to the culture medium either for 48 hours prior to irradiation. Ginseng extract showed protective effect against the formation of dsb when it was treated for 48 hours before 100 Gy gamma-ray-irradiation. While repair was almost completed until 220.2 minutes after irradiation, DNA repair of irradiated cells in the presence of ginseng extract was did not return to the corresponding control levels even after 621.8 minutes. From these data, it could be calculated that ginseng reduced the relative strand scission factor (RSSF) by about 2. Therefore, it could be concluded that ginseng has radioprotective effect against gamma-ray induced DNA dsb and repair in cultured mouse lymphocytes.

Animals↗

PubMed2000

Characterization of mammalian RAD51 double strand break repair using non-lethal dominant-negative forms.

In contrast to yeast RAD51, mammalian mRAD51 is an essential gene. Its role in double strand break (DSB) repair and its consequences on cell viability remain to be characterized precisely. Here, we used a hamster cell line carrying tandem repeat sequences with an I-SCE:I cleavage site. We characterized conservative recombination after I-SCE:I cleavage as gene conversion or intrachromatid crossing over associated with random reintegration of the excised reciprocal product. We identified two dominant-negative RAD51 forms that specifically inhibit conservative recombination: the yeast ScRAD51 or the yeast-mouse chimera SMRAD51. In contrast, the mouse MmRAD51 stimulates conservative recombination. None of these RAD51 forms affects non-conservative recombination or global DSB healing. Consistently, although resistance to gamma-rays remains unaffected, MmRAD51 stimulates whereas ScRAD51 or SMRAD51 prevents radiation-induced recombination. This suggests that mRAD51 does not significantly affect the global DSB repair efficiency but controls the classes of recombination events. Finally, both ScRAD51 and SMRAD51 drastically inhibit spontaneous recombination but not cell proliferation, showing that RAD51-dependent spontaneous and DSB-induced conservative recombination can be impaired significantly without affecting cell viability.

Animals↗