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Results for “Double stranded breaks”

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At least 703 records · Page 39Linked to original sources

PubMed2006

SUMO modification of human XRCC4 regulates its localization and function in DNA double-strand break repair.

The nonhomologous end-joining (NHEJ) pathway is responsible for rejoining the majority of double-strand breaks in mammalian cells, including the programmed breaks introduced by V(D)J recombination. The regulation of the enzymatic activities associated with this recombination pathway is still largely unknown. Here we report that human XRCC4 (for X-ray cross-complementation group 4), a protein essential for NHEJ, is subject to posttranslational protein modification. The modifier peptide, SUMO, can be added to XRCC4 both in vitro and in vivo. The site of modification is mapped to lysine 210 by using specific mutagenesis. A protein mutated such that it cannot be SUMOylated remains localized in the cytoplasm rather than accumulating in the nucleus. Cells expressing only the mutated protein are radiation sensitive and fail to complete V(D)J recombination. Genetic fusion of the SUMO sequence to the C terminus of the mutant restores nuclear localization and radiation resistance. The modification may serve a regulatory role. Our finding fits with an emerging literature associating SUMO modification with the control of the repair and recombination associated with DNA breaks.

Cell Nucleus↗

PubMed2000

Inhibition of repair of radiation-induced DNA double-strand breaks by nickel and arsenite.

The effect of arsenite or nickel on the repair of DNA double-strand breaks (DSBs) was studied in gamma-irradiated Chinese hamster ovary cells using pulsed-field gel electrophoresis. After treatment with nickel chloride or arsenite for 2 h, cells were irradiated with gamma rays at a dose of 40 Gy, and the numbers of DNA DSBs were measured immediately after irradiation as well as at 30 min postirradiation. Both arsenite and nickel(II) inhibited repair of DNA DSBs in a concentration-dependent manner; 0.08 mM arsenite significantly inhibited the rejoining of DSBs, while 76 mM nickel was necessary to observe a clear inhibition. The mean lethal concentrations for the arsenite and nickel(II) treatments were approximately 0.12 and 13 mM, respectively. This indicates that the inhibition of repair by arsenite occurred at a concentration at which appreciable cell survival occurred, but that nickel(II) inhibited repair only at cytotoxic concentrations at which the cells lost their proliferative ability. These novel observations provide insight into the mechanisms underlying the effects of combined exposure to arsenite and ionizing radiation in our environment.

Animals↗

PubMed1999

Rejoining of DNA double-strand breaks in Ku80-deficient mouse fibroblasts.

The role of Ku80 in the repair of DNA double-strand breaks (DSBs) was examined in fibroblasts derived from a Ku80 knockout mouse model described by Nussenzweig et al. (Nature 382, 551-555, 1996). Primary fibroblasts from Ku80+/+ and Ku80-/- mice were immortalized by transfection with plasmids containing either the human MYC proto-oncogene or the Simian virus 40 (SV40) T antigen and were used to measure induction and rejoining of DSBs after exposure to ionizing radiation. The number of DSBs in the cells was quantified by either asymmetric field-inversion gel electrophoresis (AFIGE) or clamped homogeneous electrical-field gel electrophoresis (CHEF). The latter method was introduced for a more reliable quantification of repair even when DNA degradation occurs in a fraction of the irradiated cell population during the postirradiation incubation time. The results confirm that Ku80-deficient mouse fibroblasts are sensitive to ionizing radiation and demonstrate that the increased radiosensitivity may result from a deficiency in DSB rejoining. The results further indicate that unless techniques are employed that allow for distinction between DNA degradation and DNA repair, erroneous conclusions may be drawn regarding the potential of cells to repair DSBs.

Animals↗

PubMed1992

Variation in radiation-induced formation of DNA double-strand breaks as a function of chromatin structure.

The influence of chromatin structure on induction of DNA double-strand breaks (DSBs) by X radiation was studied in DNA from CHO cells. Whole cells, nuclei with condensed or relaxed chromatin, and deproteinized DNA in agarose plugs were irradiated and DSB formation was measured as a decrease in the length of DNA by nondenaturing, pulsed-field, agarose gel electrophoresis. The yield of DSBs in deproteinized DNA (2.3 x 10(-10) DSBs Da-1 Gy-1) was observed to be 70 times greater than the yield of DSBs (3.1 x 10(-12) DSBs Da-1 Gy-1) observed in DNA in the intact cell nucleus. Organization of DNA into the basic nucleosome repeat structure and condensation of the chromatin fiber into higher-order structure protected DNA from DSB induction by factors of 8.3 and 4.5, respectively. An additional twofold protection of DNA in fully condensed chromatin occurred in the intact cell nucleus. Since this protection did not appear to involve chromatin structure, we speculate that this additional protection may result from the association of soluble protein and nonprotein sulfhydryls with DNA in the intact cell nucleus. The results are consistent with the organization of nuclear DNA into both basic nucleosome repeat structure and higher-order chromatin structure providing significant protection against DSB induction.

Animals↗

PubMed1997

Higher-order chromatin structure-dependent repair of DNA double-strand breaks: modeling the elution of DNA from nucleoids.

A possible relationship between the repair of DNA double-strand breaks (DSBs) and their distribution within higher-order chromatin (Johnston and Bryant, Int. J. Radiat. Biol. 66, 531-536, 1994) has recently been demonstrated. Radiosensitive cells deficient for components of the DNA-dependent protein kinase DSB repair pathway exhibited a particular failure in the rejoining of DSBs occurring as multiples within looped DNA structures. Here, a Poisson-based model of induction of DSBs and elution of DNA from residual nuclear structures is presented. By applying this model to cells of a panel of human and rodent cell lines, a mean of 1.6 Mbp for the size of the relevant looped structures was obtained. Such large chromatin structures are of the same magnitude as those observed by functional mapping of interphase and mitotic chromosome structure, nucleoid sedimentation and the "replicon clusters" apparent during DNA replication. This work supports the hypotheses that (1) such structures are critical targets for induction of DSBs and (2) the distribution of damage within these domains may be a factor in the response and sensitivity of mammalian cells to ionizing radiation.

Animals↗

PubMed1998

Misrejoining of double-strand breaks after X irradiation: relating moderate to very high doses by a Markov model.

Misrejoining of double-strand breaks (DSBs) detected with pulsed-field gel electrophoresis (PFGE) after X irradiation of human cells at very high doses (80-160 Gy) is related to dose-response relationships for chromosome aberrations at moderate doses (1-5 Gy) by the Sax-Markov binary eurejoining/misrejoining (SMBE) model. The SMBE model applies Sax's breakage-and-reunion hypothesis to a subset of DSBs active in binary misrejoining and in binary eurejoining (accidental restitution). The model is numerically consistent with both data on chromosome aberrations and the data obtained by PFGE if proximity effects (restrictions on the range of interactions of DSB free ends) are present. Proximity effects are modeled by partitioning the cell's nucleus into approximately 400 interaction sites, with two active DSB free ends capable of rejoining only if they were produced within the same site. Neglecting one-track action, the SMBE model predicts a quadratic-linear dose-response relationship for DSB misrejoining after exposure to low-LET radiation; i.e., there is a quadratic response at moderate doses which becomes linear as the dose becomes large, rather than vice versa. The linear region results because at very high doses almost all of the active DSB free ends misrejoin rather than eurejoin.

DNA↗

PubMed2000

Induction of single- and double-strand breaks in plasmid DNA by 100-1500 eV electrons.

PURPOSE: To investigate the induction of DNA strand breaks by electrons with energies ranging from 0.1 to 1.5 keV. MATERIALS AND METHODS: Dry supercoiled plasmid DNA was irradiated with electrons of energies ranging from 0.1 to 1.5 keV and the results were compared with those obtained by gamma-irradiation of the same plasmid in solution. For electron irradiation, the plasmid was deposited on a gold substrate under a controlled atmosphere to minimize contamination of the DNA film. Electron bombardments were performed under ultra-high vacuum conditions (UHV 10(-9) torr). DNA damage was detected by gel electrophoresis followed by quantitation of the DNA bands by fluorescence or by hybridization with a radioactive probe. RESULTS: Electrons with energies from 0.1 to 1.5 keV induced single, double and multiple double-strand breaks in supercoiled plasmid DNA. For equal doses, we observed a marked increase in the efficiency of induction of double- and multiple-strand breaks in supercoiled DNA as a function of electron energy. In contrast to gamma-irradiation, the formation of small DNA fragments by electrons did not seem to be related to the production of the linear form of the plasmid. CONCLUSIONS: Electrons within the energy; range of the secondary electrons generated by high-energy ionizing radiation induce single, double and multiple double-strand breaks in DNA. Problems associated with low-energy electron irradiation experiments and dose calculations in thin films are also discussed.

DNA↗

PubMed2000

The effect of dimethyl sulphoxide on the induction and repair of double-strand breaks in human cells after irradiation with gamma-rays and accelerated ions: rapid or slow repair may depend on accessibility of breaks in chromatin of different compactness.

PURPOSE: The repair of double-strand breaks (dsb) in mammalian cells is characterized by a rapid phase with a half-life of less than half an hour and a slower phase that lasts for many hours. The proportion of slow repair increase with LET and it has been suggested that the slow repair component consists of more complex damage and is more deleterious to the cells. To see if removal of OH radicals could remove part of the damage in complex dsb and make them easier to repair, human cells were irradiated in the presence of dimethyl sulphoxide (DMSO). METHODS: Induction and repair of dsb were studied by neutral elution in human VH10 cells exposed to y-rays, helium ions (mean LET 40 keV/microm) and 80 and 125 keV/microm monoenergetic nitrogen ions in the presence and absence of 2 M DMSO. RESULTS: Incubation of cells exposed to gamma-rays, 40 keV/microm helium and 80 keV/microm N ions demonstrated that scavenging of OH radicals by DMSO removed most of the rapid repair component. The response to DMSO was less marked after 125 keV/microm nitrogen ions, where about half of the rapid repair was resistant to DMSO. CONCLUSIONS: It is unlikely that the complexity of dsb is responsible for the slow repair because the removal of OH radicals did not make the breaks easier to repair. Instead, it is suggested that rapid and slow repair can be explained on the basis of how different parts of the chromatin are accessible to repair enzymes.

Cells, Cultured↗

PubMed2004

Evidence for the involvement of double-strand breaks in heat-induced cell killing.

To identify critical events associated with heat-induced cell killing, we examined foci formation of gammaH2AX (histone H2AX phosphorylated at serine 139) in heat-treated cells. This assay is known to be quite sensitive and a specific indicator for the presence of double-strand breaks. We found that the number of gammaH2AX foci increased rapidly and reached a maximum 30 minutes after heat treatment, as well as after X-ray irradiation. When cells were heated at 41.5 degrees C to 45.5 degrees C, we observed a linear increase with time in the number of gammaH2AX foci. An inflection point at 42.5 degrees C and the thermal activation energies above and below the inflection point were almost the same for cell killing and foci formation according to Arrhenius plot analysis. From these results, it is suggested that the number of gammaH2AX foci is correlated with the temperature dependence of cell killing. During periods when cells were exposed to heat, the cell cycle-dependent pattern of cell killing was the same as the cell cycle pattern of gammaH2AX foci formation. We also found that thermotolerance was due to a depression in the number of gammaH2AX foci formed after heating when the cells were pre-treated by heat. These findings suggest that cell killing might be associated with double-strand break formation via protein denaturation.

Carcinoma, Non-Small-Cell Lung↗

PubMed2002

Induction of DNA replication-mediated double strand breaks by psoralen DNA interstrand cross-links.

The effect of DNA interstrand cross-links (cross-links) on DNA replication was examined with a cell-free SV40 origin-dependent DNA replication system. A defined template DNA with a single psoralen cross-link and the SV40 origin of replication was replicated by HeLa cell-free extract in the presence of SV40 large T antigen. The psoralen cross-link inhibited DNA replication by terminating chain elongation at 1-50 nucleotides before the cross-linked sites. The termination of DNA replication by the cross-links mediated the generation of double strand breaks near the cross-linked sites. These results are the first biochemical evidence of the generation of double strand breaks by DNA replication.

Cross-Linking Reagents↗

PubMed2001

In vivo evidence for two active nuclease motifs in the double-strand break repair enzyme RexAB of Lactococcus lactis.

In bacteria, double-strand DNA break (DSB) repair involves an exonuclease/helicase (exo/hel) and a short regulatory DNA sequence (Chi) that attenuates exonuclease activity and stimulates DNA repair. Despite their key role in cell survival, these DSB repair components show surprisingly little conservation. The best-studied exo/hel, RecBCD of Escherichia coli, is composed of three subunits. In contrast, RexAB of Lactococcus lactis and exo/hel enzymes of other low-guanine-plus-cytosine branch gram-positive bacteria contain two subunits. We report that RexAB functions via a novel mechanism compared to that of the RecBCD model. Two potential nuclease motifs are present in RexAB compared with a single nuclease in RecBCD. Site-specific mutagenesis of the RexA nuclease motif abolished all nuclease activity. In contrast, the RexB nuclease motif mutants displayed strongly reduced nuclease activity but maintained Chi recognition and had a Chi-stimulated hyperrecombination phenotype. The distinct phenotypes resulting from RexA or RexB nuclease inactivation lead us to suggest that each of the identified active nuclease sites in RexAB is involved in the degradation of one DNA strand. In RecBCD, the single RecB nuclease degrades both DNA strands and is presumably positioned by RecD. The presence of two nucleases would suggest that this RecD function is dispensable in RexAB.

Amino Acid Motifs↗

PubMed2003

The Haemophilus ducreyi cytolethal distending toxin induces DNA double-strand breaks and promotes ATM-dependent activation of RhoA.

Among bacterial protein toxins, the cytolethal distending toxins (CDTs) are unique in their ability to activate the DNA damage checkpoint responses, causing cell cycle arrest or apoptosis in intoxicated cells. We provide direct evidence that natural intoxication of cells with the Haemophilus ducreyi CDT (HdCDT) holotoxin induces DNA double-strand breaks similarly to ionizing radiation. Upon DNA damage, epithelial cells and fibroblasts promote the formation of actin stress fibres via activation of the small GTPase RhoA. This phenomenon is not toxin specific, but is part of the ATM-induced cellular responses to genotoxic stresses, including ionizing radiation. Activation of RhoA is associated with prolonged cell survival, as HdCDT-treated epithelial cells expressing a dominant-negative form of RhoA detach and consequently die faster than cells expressing a functional RhoA. Our data highlight several novel aspects of CDT biology: (i) we show that a member of the CDT family causes DNA double-strand breaks in naturally intoxicated cells, acting as a true genotoxic agent; and (ii) we disclose the existence of a novel signalling pathway for intracellularly triggered activation of the RhoA GTPase via the ATM kinase in response to DNA damage, possibly required to prolong cell survival.

Actins↗

PubMed2005

The AtRAD51C gene is required for normal meiotic chromosome synapsis and double-stranded break repair in Arabidopsis.

Meiotic prophase I is a complex process involving homologous chromosome (homolog) pairing, synapsis, and recombination. The budding yeast (Saccharomyces cerevisiae) RAD51 gene is known to be important for recombination and DNA repair in the mitotic cell cycle. In addition, RAD51 is required for meiosis and its Arabidopsis (Arabidopsis thaliana) ortholog is important for normal meiotic homolog pairing, synapsis, and repair of double-stranded breaks. In vertebrate cell cultures, the RAD51 paralog RAD51C is also important for mitotic homologous recombination and maintenance of genome integrity. However, the function of RAD51C in meiosis is not well understood. Here we describe the identification and analysis of a mutation in the Arabidopsis RAD51C ortholog, AtRAD51C. Although the atrad51c-1 mutant has normal vegetative and flower development and has no detectable abnormality in mitosis, it is completely male and female sterile. During early meiosis, homologous chromosomes in atrad51c-1 fail to undergo synapsis and become severely fragmented. In addition, analysis of the atrad51c-1 atspo11-1 double mutant showed that fragmentation was nearly completely suppressed by the atspo11-1 mutation, indicating that the fragmentation largely represents a defect in processing double-stranded breaks generated by AtSPO11-1. Fluorescence in situ hybridization experiments suggest that homolog juxtaposition might also be abnormal in atrad51c-1 meiocytes. These results demonstrate that AtRAD51C is essential for normal meiosis and is probably required for homologous synapsis.

Arabidopsis↗

PubMed2005

Detection of DNA double-strand breaks and chromosome translocations using ligation-mediated PCR and inverse PCR.

Current techniques for examining the global creation and repair of DNA double-strand breaks are restricted in their sensitivity, and such techniques mask any site-dependent variations in breakage and repair rate or fidelity. We present here a system for analyzing the fate of documented DNA breaks, using the MLL gene as an example, through application of ligation-mediated PCR. Here, a simple asymmetric double-stranded DNA adapter molecule is ligated to experimentally induced DNA breaks and subjected to seminested PCR using adapter and gene-specific primers. The rate of appearance and loss of specific PCR products allows detection of both the break and its repair. Using the additional technique of inverse PCR, the presence of misrepaired products (translocations) can be detected at the same site, providing information on the fidelity of the ligation reaction in intact cells. Such techniques may be adapted for the analysis of DNA breaks introduced into any identifiable genomic location.

Animals↗

PubMed2006

Detection of DNA double-strand breaks and chromosome translocations using ligation-mediated PCR and inverse PCR.

Cells, Cultured↗

PubMed1993

Estimation of double-strand break quality based on track-structure calculations.

The aim of this study was to investigate whether double-strand break (DSB) quality is related to the complexity of radiation-induced damage or to the spatial distribution of initial DSBs. The analysis was based on track structure calculations and DNA damage modelling. The results obtained indicate that the quality of DSBs is related to their initial spatial distribution rather than to their complexity.

DNA Damage↗

PubMed1994

Mechanisms of induction and repair of DNA double-strand breaks by ionizing radiation: some contradictions.

The various aspects of formation and repair of radiation-induced double-strand breaks (DSB) are summarized. Concerning the structure of DSB found in irradiated cells, enzymatic and microdosimetric analysis hints at complex damage of the DNA structure at the position of a DSB. With increasing LET, the DSB damage may be more complex than that induced by low-LET irradiation. Most of the DSB are repaired in the irradiated cell; apparently the kinetics of DSB repair and the fraction of unrejoined DSB determine cell survival or cell death. We do not know the details of the complex machinery of DSB repair; certainly recombination processes are involved, but there are still contradictions between our current knowledge about the mechanisms of recombinational DSB repair and the observed kinetics.

Cell Survival↗

PubMed2006

Genetic dissection of vertebrate 53BP1: a major role in non-homologous end joining of DNA double strand breaks.

53BP1 (p53 binding protein) is a BRCT domain-containing protein that is rapidly recruited to DNA double strand breaks (DSBs). To investigate the role of 53BP1 in the DNA damage response, we generated 53BP1(-/-) cells from the chicken DT40 cell line. As in mammalian cells, mutation of 53BP1 increased cellular sensitivity to ionizing radiation. Although depletion of 53BP1 resulted in checkpoint defects in mammalian cells, DT40 53BP1(-/-) cells had normal intra S phase and G2/M checkpoints. G1 specific radiosensitivity and a higher sensitivity to topoisomerase II suggested defective non-homologous end joining (NHEJ) defects in DT40 53BP1(-/-) cells. Genetic analyses confirm this suggestion as we have demonstrated an epistatic relationship between 53BP1 and the NHEJ genes, Ku70 and Artemis, but not with Rad54, a gene essential for repair of DSBs by homologous recombination. We conclude that the major role of 53BP1 in supporting survival of DT40 cells that have suffered DNA DSBs is in facilitating repair by NHEJ.

Animals↗