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At least 325 records · Page 18Linked to original sources

PubMed2005

Cell cycle-dependent regulation of double-strand break repair: a role for the CDK.

DNA Double-Strand Breaks (DSBs) are dangerous lesions that can lead to genomic instability and to cell death. Eukaryotic cells repair DSBs either by nonhomologous end joining (NHEJ) or by homologous recombination (HR). Recent work has allowed to study the ability of yeast cells to repair a single, chromosomal DSB, at different stages of the cell cycle. Yeast cells repair the broken chromosome during the G(1) stage only by NHEJ, whereas HR is the mechanism of choice during the rest of the cell cycle. HR does not require duplicated chromatids or passage through S-phase. Control over the fate of the broken chromosome is exerted by Clb-CDK activity, which is required to carry out the first step of HR, ssDNA resection. Similar results in other organisms suggest that this control is a conserved feature in all eukaryotes.

Cell Cycle↗

PubMed1990

A kinetic analysis of the repair of radiation-induced DNA double-strand breaks.

The repair of radiation-induced DNA double-strand breaks (DSBs) is analyzed kinetically. It is assumed that a fraction of the damaged sites in the DNA duplex are irreparable. The kinetic model takes the effect of radiation dose into account. The analysis of the available experimental data reveals that, although the number of irreparable DSBs is a quadratic function of radiation dose, the normalized number of irreparable DSBs correlates linearly with this variable.

DNA↗

PubMed1994

Differential repair of radiation-induced DNA damage in cells of human squamous cell carcinoma and the effect of caffeine and cysteamine on induction and repair of DNA double-strand breaks.

The goal of these experiments was to investigate further the relationship between DNA double-strand breaks and cell killing in human tumor cells, first by comparing different cell lines, and second by radiomodification studies. Field-inversion gel electrophoresis was used to quantify double-strand breaks. Two subclones of the radioresistant human squamous cell carcinoma line SQ20B (SQD9 and SQG6) were compared. These subclones differed in DNA index by a factor of 1.7 but showed the same resistance to radiation as cells of the parental cell line. It was found that, although induction of DSBs was not significantly different in the two cell lines, the t1/2 of the fast component of repair was significantly shorter for SQD9 cells, leading to greater overall repair which was not reflected in increased survival. Caffeine and cysteamine were tested as modifiers of radiosensitivity, using the radioresistant SQ20B line and the radiosensitive SCC61 cell line. No effect of caffeine was seen when the drug was present only during irradiation. Postirradiation incubations with caffeine, however, resulted in a dose reduction factor greater than 2.0 in cell survival for both cell lines. In contrast, induction of DSBs was reduced by caffeine, and no effect on DSB repair was observed. Cysteamine led to a dose protection factor greater than 1.8 in cell survival in both cell lines. A reduction in induced DSBs was found at high doses corresponding approximately with the increase in cell survival. Over the same (low) dose range, however, the correlation between DSB induction and cell killing was poor. These data indicate that DSB induction does not correlate well with cell killing either for different cell lines, for radiochemical modification (cysteamine) or for some other types of modification (caffeine).

Caffeine↗

PubMed1996

The joining of non-complementary DNA double-strand breaks by mammalian extracts.

We have developed a high efficiency system in which mammalian extracts join DNA double-strand breaks with non-complementary termini. This system has been used to obtain a large number of junction sequences from a range of different break-end combinations, allowing the elucidation of the joining mechanisms. Using an extract of calf thymus it was found that the major mechanism of joining was by blunt-end ligation following removal or fill-in of the single-stranded bases. However, some break-end combinations were joined through an efficient mechanism using short repeat sequences and we have succeeded in separating this mechanism from blunt-end joining by the biochemical fractionation of extracts. Characterization of activities and sequence data in an extensively purified fraction that will join ends by the repeat mechanism led to a model where joining is initiated by 3' strand invasion followed by pairing to short repeat sequences close to the break site. Thus the joining of double-strand breaks by mammalian extracts is achieved by several mechanisms and this system will allow the purification of the factors involved in each by the judicial choice of the non-complementary ends used in the assay.

Animals↗

PubMed2001

ATM phosphorylates histone H2AX in response to DNA double-strand breaks.

A very early step in the response of mammalian cells to DNA double-strand breaks is the phosphorylation of histone H2AX at serine 139 at the sites of DNA damage. Although the phosphatidylinositol 3-kinases, DNA-PK (DNA-dependent protein kinase), ATM (ataxia telangiectasia mutated), and ATR (ATM and Rad3-related), have all been implicated in H2AX phosphorylation, the specific kinase involved has not yet been identified. To definitively identify the specific kinase(s) that phosphorylates H2AX in vivo, we have utilized DNA-PKcs-/- and Atm-/- cell lines and mouse embryonic fibroblasts. We find that H2AX phosphorylation and nuclear focus formation are normal in DNA-PKcs-/- cells and severely compromised in Atm-/- cells. We also find that ATM can phosphorylate H2AX in vitro and that ectopic expression of ATM in Atm-/- fibroblasts restores H2AX phosphorylation in vivo. The minimal H2AX phosphorylation in Atm-/- fibroblasts can be abolished by low concentrations of wortmannin suggesting that DNA-PK, rather than ATR, is responsible for low levels of H2AX phosphorylation in the absence of ATM. Our results clearly establish ATM as the major kinase involved in the phosphorylation of H2AX and suggest that ATM is one of the earliest kinases to be activated in the cellular response to double-strand breaks.

Androstadienes↗

PubMed1994

Increased incidence of DNA double-strand breaks and anti-ds DNA antibodies in blood of workers occupationally exposed to asbestos.

Asbestos, proven to be carcinogenic in humans and animals, is reported to have no genotoxic effect. Asbestos workers have an increased risk of lung cancer, mesothelioma, and other tumours. Earlier findings showed that crocidolite can induce DNA strand breaks in cultured rat embryo cells as assessed by nick translation. We investigated DNA double-strand breaks in white blood cells (WBC) of ten workers occupationally exposed to asbestos. According to our results, obtained with neutral filter elution, individuals who had been exposed to asbestos fibres showed two to four times more DNA double-strand breaks (dsb) in white blood cells than ten non-exposed persons. The induced DNA fragments are of about 250 kb (compared to chromosomal DNA of Saccharomyces cerevisiae standard marker). Using additionally the chromosomal DNA protective method of agarose-plugs, DNA fragments in the range of 200 to 1000 kb have been found in the white blood cells of the same ten workers occupationally exposed to asbestos. In the white blood cells of non-exposed subjects no DNA fragments could be detected with this method. Compared to 51 non-exposed persons, elevated anti-ds DNA antibody concentrations were found in ten workers occupationally exposed to asbestos. The fact that workers occupationally exposed to asbestos have distinctly more double-strand breaks and anti-ds DNA antibodies could mean that an increased incidence of DNA-fragments may be an important indicator in the chronic effect of asbestos-associated carcinogenesis. Apparently, the chronic effects of asbestos observed here do not seem to be identical with that of previously reported acute in vitro effects.

Adult↗

PubMed1989

Sensitivity of Chinese hamster ovary mutants defective in DNA double strand break repair to topoisomerase II inhibitors.

xrs-1 is an ionizing radiation sensitive Chinese hamster ovary (CHO) strain and has a defect in double strand break rejoining. It is also highly sensitive to topoisomerase II inhibiting anticancer drugs. Sensitivity is specific for those drugs that inhibit the breakage/reunion mechanism of topoisomerase II. xrs-1 and its parent strain CHO-K1 have equal levels of topoisomerase II activity, assayed by their ability to unknot complex knotted P4 head DNA. Drug stimulated protein-DNA complex formation was similar in xrs-1 and CHO-K1, showing that they accrued equal levels of drug induced lesions. Thus sensitivity most likely results from subsequent differences in the processing of these lesions rather than the rate of formation. Drug sensitivity is directly related to the xrs phenotype since drug and gamma-ray resistance are coordinately reactivated by azacytidine treatment. All six members of the xrs complementation group are hypersensitive to etoposide. Sensitivity is not a feature common to all X-ray sensitive mutants but is shown by another complementation group, which also has a defect in double strand break rejoining. These results thus demonstrate a correlation between an inability to rejoin double strand breaks and sensitivity to topoisomerase II targeting antitumor drugs.

Animals↗

PubMed1998

Biotin-labeled hairpin oligonucleotides: probes to detect double-strand breaks in DNA in apoptotic cells.

Hairpin oligonucleotides were synthesized with stems ending in a double-stranded structure, which can be ligated to double-strand breaks in DNA, and with loops that contain nucleotides modified by the attachment of biotin. These probes specifically and sensitively detect double-strand breaks in apoptotic cells. Localization of these probes is restricted to areas of chromatin characteristic of apoptosis, whereas much more diffuse labeling was obtained when all available 3' DNA ends were labeled by terminal transferase. In principle, hairpin oligonucleotide probes can be designed with any type of 3' or 5' overhang complementary to double-strand DNA termini being detected.

Animals↗

PubMed1984

Formation and rejoining of deoxyribonucleic acid double-strand breaks induced in isolated cell nuclei by antineoplastic intercalating agents.

The biochemical characteristics of the formation and disappearance of intercalator-induced DNA double-strand breaks (DSB) were studied in nuclei from mouse leukemia L1210 cells by using filter elution methodology [Bradley, M. O., & Kohn, K.W. (1979) Nucleic Acids Res. 7, 793-804]. The three intercalators used were 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA), 5-iminodaunorubicin (5-ID), and ellipticine. These compounds differ in that they produced predominantly DNA single-strand breaks (SSB) (m-AMSA) or predominantly DNA double-strand breaks (ellipticine) or a mixture of both SSB and DSB (5-ID) in whole cells. In isolated nuclei, each intercalator produced DSB at a frequency comparable to that which is produced in whole cells. Moreover, these DNA breaks reversed within 30 min after drug removal. It thus appeared that neither ATP nor other nucleotides were necessary for intercalator-dependent DNA nicking-closing reactions. The formation of the intercalator-induced DSB was reduced at ice temperature. Break formation was also reduced in the absence of magnesium, at a pH above 6.4 and at NaCl concentrations above 200 mM. In the presence of ATP and ATP analogues, the intercalator-induced cleavage was enhanced. These results suggest that the intercalator-induced DSB are enzymatically mediated and that the enzymes involved in these reactions can catalyze DNA double-strand cleavage and rejoining in the absence of ATP, although the occupancy of an ATP binding site might convert the enzyme to a form more reactive to intercalators. Three inhibitors of DNA topoisomerase II--novobiocin, nalidixic acid, and norfloxacin--reduced the formation of DNA strand breaks.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1995

Chromatin structure and radiation-induced DNA strand breaks in human cells: soluble scavengers and DNA-bound proteins offer a better protection against single- than double-strand breaks.

In the present investigation we have studied the formation of DNA double-strand breaks (dsbs) in different chromatin substrates and made a comparison with our previous results on radiation-induced single-strand breaks (ssb). Removal of soluble scavengers increased the number of ssbs by a factor of 2, but increased dsbs only 1.2 times. Decondensation of the chromatin increased ssbs and dsbs to the same extent, by a factor of 3-4. Removal of DNA-bound proteins gave an additional increase in ssbs and dsbs by a factor of 14 and 5 respectively. The increase in both ssbs and dsbs was caused by OH radicals. The differential effect on ssbs and dsbs can be explained by assuming that dsbs are formed mainly by multiple hits from clusters of ionizations close to the DNA. When the DNA-bound proteins are removed, more distant radicals can reach the DNA, which favours ssb formation. Under those conditions dsbs also induced by single OH radicals become important. DNA-bound proteins protect DNA only to a limited extent by acting as scavengers of OH radicals. The largest part of the protection depends on the fact that DNA in the chromatin is made up of large, compact aggregates, where the distance between separate aggregates exceeds the effective range of the OH radicals and the amount of water inside the aggregates is too small to give a maximal contribution of OH radicals compared with when DNA is more evenly distributed in a given volume.

Cells, Cultured↗

PubMed1988

Vector-mediated DNA double-strand break repair analysis in normal, and radiation-sensitive, Chinese hamster V79 cells.

DNA double-strand break repair was assessed in 2 new radiation-sensitive V79 hamster cell lines (irs1 and irs2) by their ability to rejoin restriction endonuclease cuts in a transferred selectable SV40--E. coli gpt recombinant gene. The studied gene was carried in the vector pPMH16 which also contained a second selectable HSVtk-neo recombinant gene which acted as a control for DNA transformation. The parental V79 cells showed correct rejoining of KpnI and EcoRV double-strand breaks in approximately 18% and 36% of transformants respectively (correcting for the expression of undamaged gpt in neo+ transformants). irs1 shows a significantly reduced (approximately 3-fold) ability to rejoin correctly such double-strand scissions. However, irs2 rejoined such lesions as correctly as the V79 cells. The data are discussed in the context of the assay and the possible repair deficiencies of these radiosensitive mutant cells.

Animals↗

PubMed2006

DNA double-strand break repair: all's well that ends well.

Breaks in both DNA strands are a particularly dangerous threat to genome stability. At a DNA double-strand break (DSB), potentially lost sequence information cannot be recovered from the same DNA molecule. However, simple repair by joining two broken ends, though inherently error prone, is preferable to leaving ends broken and capable of causing genome rearrangements. To avoid DSB-induced genetic disinformation and disruption of vital processes, such as replication and transcription, cells possess robust mechanisms to repair DSBs. Because all breaks are not created equal, the particular repair mechanism used depends largely on what is possible and needed based on the structure of the broken DNA. We argue that although categorizing different DSB repair mechanisms along pathways and subpathways can be conceptually useful, in cells flexible and reversible interactions among DSB repair factors form a web from which a nonpredetermined path to repair for any number of different DNA breaks will emerge.

Animals↗

PubMedDate not supplied

[Effect of single-stranded and double-stranded breaks on the melting temperature of phage T2 DNA].

The effect of single- and double-stranded breaks in DNA phage T2, on the melting temperature of this DNA in the 0,05 M SSC solution, was investigated. The number of cleavages per 1000 nucleotide pairs varied in the range of 0 to 10. It is shown that single- and double-stranded breaks affect the melting temperature with approximately (within 20%) the same efficiency. The relationship between the melting temperature shift (delta Tm) and the number of cleavages is non-linear. The magnitude of the effect is characterized by delta Tm of 2 +/- 0.4 degrees C for the average inter-cleavage distance of 200 base pairs. It is shown that the observed melting curves are non-equilibrium ones, which is probably due to the fact that the effect of cleavages on the melting temperature is largely results from the complete and practically irreversible separation of strands.

Coliphages↗

PubMed1999

Visual quantification of DNA double-strand breaks in bacteria.

In this paper, we describe a method for the visualization of double-strand breaks in a single electrostretched Escherichia coli DNA molecule. We also provide evidence that electrostretched or migrated DNA under neutral microgel electrophoresis conditions is made up of individual chromosomes. Using the neutral microgel electrophoresis technique, DNA migration (stretching) was measured and the number of DNA double-strand breaks were counted following exposure of E. coli cells to 0, 12.5, 25, 50, or 100 rad of X-rays. The use of an intense fluorescent dye, YOYO and custom-made slides have helped us in visualizing individual bacterial DNA molecules. Bacterial DNA appears similar in structure compared to electrostretched DNA from human lymphocytes. We were able to detect changes in DNA migration (stretching) induced by an X-ray dose as low as 12.5 rad and an increase in the number of DNA breaks induced by a dose as low as 25 rad. The extent of DNA migration and number of breaks were directly correlated to X-ray dosage.

Benzoxazoles↗

PubMed1993

The significance of DNA double-strand breaks in the UV inactivation of yeast cells.

The radiation-sensitive mutant rad 54-3 of Saccharomyces cerevisiae temperature-conditional for the repair of DNA double-strand breaks was exposed to 254 nm ultraviolet radiation and incubated at the restrictive and permissive temperatures. A large difference in survival was seen indicating the involvement of double-strand breaks in cellular inactivation at least in this strain. Pulsed-field gel electrophoresis of DNA showed that double-strand breaks are not directly induced but develop upon incubation under growth conditions. Their number is highest after about 4 h, after 8 h repair is complete in wild-type cells. With the aid of the excision-deficient double mutant rad3rad54 it could be demonstrated that strand break formation proceeds independent of excision repair.

DNA Damage↗

PubMed1987

Promotion of double-strand break repair by human nuclear extracts preferentially involves recombination with intact homologous DNA.

Parameters of DNA double strand break (dsb) repair catalysed by human nuclear extract were analysed using, as substrate, the replicative form (RF) of M13 mp8 in which a single double strand break (dsb) was introduced by restriction. After incubation with the extract, the dsb repair was estimated by the ability of the incubated RF to produce plaques following transfection into JM 109 (Rec A-) bacteria. The possibility of recombination with a purified fragment from M13 mp8 RF enhances up to 20 times the plaquing ability of the RF. The repair by recombination occurs under several conditions: i) the break in the RF must be located in the region of homology with the fragment. ii) the fragment has to be intact in the region corresponding to the break in the RF. iii) a minimal length of homology between the region surrounding the dsb in the RF, and the fragment is required. The in vitro reaction is ATP dependent and dNTP's partially dependent. Dephosphorylation of the free ends in the RF decreases the repair by ligation but is without effect on the recombination.

Cell Nucleus↗

PubMed1996

Double strand breaks induced by low doses of gamma rays or heavy ions: quantitation in nonradioactive human DNA.

We have developed a method of quantitating low frequencies (0-30 sites/10(9) base pairs) of double strand breaks in approximately 1 microgram of nonradioactive human DNA. Unirradiated or irradiated DNA is digested with the restriction endonuclease NotI, producing cleavage fragments that include a major group centered at approximately 1.2-1.3 Mbp. The DNA molecules are separated as a function of size by transverse alternating field electrophoresis. The frequency of double strand breaks is computed directly from the decrease in number average molecular length induced in the 1.2- to 1. 3-Mbp cleavage fragment group by 137Cs gamma or Fe26+ (1.1 GeV/nucleon) irradiation vs the corresponding unirradiated DNA samples. The double strand break frequency can be quantitated easily in the dose range of 0-10 cGy of gamma rays. The frequency of breaks per unit dose calculated for gamma irradiation of DNA in human cells (approximately 4.6 double strand breaks/10(9) bp/Gy) is within the range of values obtained by others (2-8 sites/10(9) bp/Gy) who used methods requiring higher doses.

Cell Line, Transformed↗

PubMed1985

Effect of double-strand breaks on homologous recombination in mammalian cells and extracts.

We examined the effect of double-strand breaks on homologous recombination between two plasmids in human cells and in nuclear extracts prepared from human and rodent cells. Two pSV2neo plasmids containing nonreverting, nonoverlapping deletions were cotransfected into cells or incubated with cell extracts. Generation of intact neo genes was monitored by the ability of the DNA to confer G418r to cells or Neor to bacteria. We show that double-strand breaks at the sites of the deletions enhanced recombination frequency, whereas breaks outside the neo gene had no effect. Examination of the plasmids obtained from experiments involving the cell extracts revealed that gene conversion events play an important role in the generation of plasmids containing intact neo genes. Studies with plasmids carrying multiple polymorphic genetic markers revealed that markers located within 1,000 base pairs could be readily coconverted. The frequency of coconversion decreased with increasing distance between the markers. The plasmids we constructed along with the in vitro system should permit a detailed analysis of homologous recombinational events mediated by mammalian enzymes.

Animals↗