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Results for “Double stranded breaks”

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PubMed1993

Application of programmable, autonomously controlled electrode (PACE) technology to the development of an improved pulsed field gel electrophoresis assay for DNA double-strand breaks in mammalian cells.

PACE (programmable, autonomously controlled electrodes) pulsed field gel electrophoresis technology was used to develop a DNA double-strand break assay that simultaneously combined (1) resolution across a broad range of DNA sizes, (2) high sensitivity (in terms of detecting dsb at low doses) and (3) speed. A 48 h PACE/dsb assay resolves DNA fragments ranging in size from 0.2 to 6 Mb, and detects damage induced by as little as 2 Gy of gamma-radiation. A different set of electrophoretic conditions resolves DNA between 1.5 and 6 megabases in 23 h, with a detection limit of about 5 Gy. A third electrophoretic protocol, while not resolving DNA fragments greater than 50 kb in length, detects DNA dsb in CHO cells induced by 15 Gy or more of gamma-rays after only a 1 h run. In particular, the 48 h PACE/dsb assay should prove useful in studies aimed at understanding the mechanisms whereby a variety of biological, chemical and physical agents induce DNA dsb in eukaryotes.

Animals↗

PubMed1996

Rejoining of DNA double-strand breaks in X-irradiated CHO cells studied by constant- and graded-field gel electrophoresis.

Induction and repair of double-strand breaks (dsb) were measured in exponentially growing CHO-10A cells using the constant- and graded-field gel electrophoresis. Dsb repair was studied after an X-ray dose of 60 Gy. The repair curve obtained was biphasic with the respective half-times of tau 1 = 3.8 +/- 0.9 and tau 2 = 118 +/- 30 min. The number of non-reparable dsb was measured for X-ray doses up to 180 Gy and was found to be only a small fraction (14%) of all non-rejoinable breaks determined previously using the alkaline unwinding technique. The ratio of non-reparable dsb to the number of lethal events calculated from survival curves is 0.14:1. This result indicates that for CHO cells nonreparable dsb represent only a small fraction of lethal damage. This is in line with the cytogenetic observation that cell killing mainly results from mis-rejoined events (i.e. exchange aberrations, translocations, interstitial deletions). The kinetics of dsb rejoining were found to be independent of the size of the fragments involved (between 1 and 10 Mbp). In addition, the rejoining kinetics of DNA fragments < or = 1 Mbp did not show the formation of new DNA fragments with time after irradiation indicating the absence of programmed cell death in irradiated CHO cells.

Animals↗

PubMed1997

Heat effects on the repair of DNA double-strand breaks in CHO cells.

The effect of heat (43-45 degrees C) on the induction and the repair of DNA double-strand breaks (dsb) was studied in CHO cells after 60 Gy of X-rays using constant-field gel electrophoresis. Heat given prior to irradiation was found not to alter the number of dsb when measured immediately after irradiation. In non-heated cells, about 80% of all dsb were rapidly repaired with a half-time of 4 min, while 20% were repaired more slowly with T4 = 160 min. These kinetics were grossly altered by heat. Both the fast and the slow process were retarded. However, the main effect of heat was an increase in the number of slowly rejoined dsb. This increase was shown to result from the additional formation (up to 1.4-fold the initial number) of dsb early during the repair course. It is suggested that the additional dsb arose from base damage, the repair of which was unbalanced by heat. No evidence was found for apoptosis being involved in this process. The kinetics of the additional dsb was found to correlate with thermal radiosensitization.

Animals↗

PubMed2000

Exo1 roles for repair of DNA double-strand breaks and meiotic crossing over in Saccharomyces cerevisiae.

The MRE11, RAD50, and XRS2 genes of Saccharomyces cerevisiae are involved in the repair of DNA double-strand breaks (DSBs) produced by ionizing radiation and by radiomimetic chemicals such as methyl methanesulfonate (MMS). In these mutants, single-strand DNA degradation in a 5' to 3' direction from DSB ends is reduced. Multiple copies of the EXO1 gene, encoding a 5' to 3' double-strand DNA exonuclease, were found to suppress the high MMS sensitivity of these mutants. The exo1 single mutant shows weak MMS sensitivity. When an exo1 mutation is combined with an mre11 mutation, both repair of MMS-induced damage and processing of DSBs are more severely reduced than in either single mutant, suggesting that Exo1 and Mre11 function independently in DSB processing. During meiosis, transcription of the EXO1 gene is highly induced. In meiotic cells, the exo1 mutation reduces the processing of DSBs and the frequency of crossing over, but not the frequency of gene conversion. These results suggest that Exo1 functions in the processing of DSB ends and in meiotic crossing over.

Crossing Over, Genetic↗

PubMed2002

Incorporation of large heterologies into heteroduplex DNA during double-strand-break repair in mouse cells.

In this study, the formation and repair of large (>1 kb) insertion/deletion (I/D) heterologies during double-strand-break repair (DSBR) was investigated using a gene-targeting assay that permits efficient recovery of sequence insertion events at the haploid chromosomal immunoglobulin (Ig) mu-locus in mouse hybridoma cells. The results revealed that (i) large I/D heterologies were generated on one or both sides of the DSB and, in some cases, formed symmetrically in both homology regions; (ii) large I/D heterologies did not negatively affect the gene targeting frequency; and (iii) prior to DNA replication, the large I/D heterologies were rectified.

Animals↗

PubMed2004

DNA double strand break repair in human bladder cancer is error prone and involves microhomology-associated end-joining.

In human cells DNA double strand breaks (DSBs) can be repaired by the non-homologous end-joining (NHEJ) pathway. In a background of NHEJ deficiency, DSBs with mismatched ends can be joined by an error-prone mechanism involving joining between regions of nucleotide microhomology. The majority of joins formed from a DSB with partially incompatible 3' overhangs by cell-free extracts from human glioblastoma (MO59K) and urothelial (NHU) cell lines were accurate and produced by the overlap/fill-in of mismatched termini by NHEJ. However, repair of DSBs by extracts using tissue from four high-grade bladder carcinomas resulted in no accurate join formation. Junctions were formed by the non-random deletion of terminal nucleotides and showed a preference for annealing at a microhomology of 8 nt buried within the DNA substrate; this process was not dependent on functional Ku70, DNA-PK or XRCC4. Junctions were repaired in the same manner in MO59K extracts in which accurate NHEJ was inactivated by inhibition of Ku70 or DNA-PK(cs). These data indicate that bladder tumour extracts are unable to perform accurate NHEJ such that error-prone joining predominates. Therefore, in high-grade tumours mismatched DSBs are repaired by a highly mutagenic, microhomology-mediated, alternative end-joining pathway, a process that may contribute to genomic instability observed in bladder cancer.

Antigens, Nuclear↗

PubMed2002

The fraction of DNA released on pulsed-field gel electrophoresis gels may differ significantly between genomes at low levels of double-strand breaks.

A common way to use pulsed-field gel electrophoresis (PFGE) for measuring the induction and repair of DNA double-strand breaks (DSBs) in mammalian cells is by using the fraction of total DNA released, FR, from the plug. We have analyzed the general relationship between initial chromosome sizes and FR. It is shown that, because of the difference in initial chromosomal size, the discrepancy in FR values between human and rodent cells may become significant at doses of radiation producing approximately 5 DSBs/100 Mbp or less.

Animals↗

PubMed1991

Comparison of repair of DNA double-strand breaks caused by neutron or gamma radiation in cultured human cells.

The dose-response for the induction of initial double-strand breaks (dsb) in DNA of human epithelioid cells by JANUS 0.85 MeV fission-spectrum neutrons was parabolic as assayed by a calibrated neutral filter elution technique. The relative biological effectiveness (RBE) of these neutrons relative to 60Co gamma-rays was unity. The kinetics of repair after a 60 Gy gamma-ray exposure were biphasic. About 65% of these dsb were rapidly repaired (T 1/2 of approximately 2 min), and the remainder were almost completely removed after 150 min at a slower rate (T 1/2 = 30 min). After the same dose of JANUS neutrons, the rapid repair component was markedly reduced (possibly not a significant repair component), and the bulk of the dsb were sealed more slowly (T 1/2 = 90 min). After 150 min, 25% remained unsealed. Even after a lower neutron dose (20 Gy), a proportion of the dsb were refractory to repair. Thus, unrepaired (or irreparable) dsb induced by high energy neutrons might explain the high RBE of neutrons for cell killing.

Cell Line↗

PubMed1990

Physical basis for detection of DNA double-strand breaks using neutral filter elution.

Results using neutral filter elution are difficult to explain if this method detects only DNA double-strand breaks (DSBs). In an attempt to understand neutral filter elution, the size of DNA pieces eluted from filters was measured using pulsed-field gel electrophoresis. Contrary to expectation, the size of the pieces was independent of radiation dose and time of elution, and much smaller (approximately 460 kb) than anticipated based on the expected number of DSBs induced. Shearing of the DNA molecule, the presence of nonspecific nucleases, and the influence of DNA-associated proteins were examined but could not explain our results. Consequently, we propose that cell lysis causes swelling of the DNA gel, and the exposed fraction of DNA on the surface of the gel is then sheared as the elution solution flows through the filter. We suggest that the rate of DNA elution measured using neutral filter elution is dependent upon the number of DSBs present, the composition of the eluting solution, especially with regard to the presence of molecules which can influence chromatin swelling on the filter, and the conformation or "packaging" of DNA before lysis.

Animals↗

PubMed2003

Induction of DNA double strand breaks by arsenite: comparative studies with DNA breaks induced by X-rays.

Chinese hamster ovary (CHO-K1) cell line and two of its DNA double strand break (DSB) repair deficient mutant cell lines, xrs-5 (Ku80 mutant) and irs-20 (DNA-PKcs mutant), were treated with various concentrations of sodium arsenite for 2.5h, and the colony forming abilities were studied. The wild type cells showed the highest cell survival, while xrs-5 cells showed the lowest survival, and irs-20 cells had an intermediate survival. These results are very similar to the cell survival curves induced by X-rays in these three cell lines. Our data also show the dose dependent induction of DNA-DSBs in these cell lines exposed to arsenite. However, in order to obtain a similar cell survival in wild type cells, twice as many DNA-DSBs are necessary with arsenite exposure when compared with X-rays, suggesting that the types of DNA lesions leading to DSB induced by arsenite are different from those by X-rays. Based on these data, further mechanistic investigations including the involvement of DNA-DSB repair proteins are warranted in the recovery process from arsenic (As) exposure.

Animals↗

PubMed1987

Single- and double-strand break formation in DNA irradiated in aqueous solution: dependence on dose and OH radical scavenger concentration.

The yields of single- and double-strand breaks (SSB and DSB) in calf thymus DNA, after 60Co gamma irradiation in dilute aqueous solution, have been determined via molecular weight measurements using a low-angle laser light scattering technique. The irradiations were administered to N2O-containing solutions of DNA in the absence and presence of oxygen and with different concentrations of the OH radical scavengers phenol, tertiary butanol, and methanol. OH radicals were found to produce SSB linearly with dose with a G value of 55 nmol J-1 and 54 nmol J-1 in deoxygenated and oxygenated solutions, respectively. DSB were formed according to a linear-quadratic dose relationship and the G value of linearly formed DSB were GDSB alpha(r.t.) = 3.5 nmol J-1 in deoxygenated and 3.2 nmol J-1 in oxygenated solution. The ratio of GSSB/GDSB alpha(r.t.) = gamma of 19 +/- 6 was independent of the scavenger concentration in the case of tertiary butanol and methanol-containing solutions. GDSB alpha(r.t.) is interpreted to result from a radical site transferred from a sugar moiety of the cleaved strand to the complementary intact strand. This process of radical transfer and subsequent cleavage of the second strand occurs with a probability of about 6 +/- 2% in the presence of oxygen at all scavenger concentrations studied. These data on scavenging capacity on GDSB alpha(r.t.) suggest that the double-strand breakage produced via radical transfer remains higher than that resulting from direct effect, up to scavenging capacities of about 10(9) s-1.

Butanols↗

PubMed2006

Double-strand break repair in bacteriophage T4: coordination of DNA ends and effects of mutations in recombinational genes.

Coordination of DNA ends during double-strand break (DSB) repair was studied in crosses of bacteriophage T4 in which DSBs were induced site-specifically by SegC endonuclease in the DNA of only one of the parents. Coupling of the genetic exchanges to the left and to the right of the DSB was measured in the wild-type genetic background as well as in T4 strains bearing mutations in several recombination genes: 47, uvsX, uvsW, 59, 39 and 61. The observed quantitative correlation between the degree of coupling and position of the recombining markers in relation to the DSB point implies that the two variants of the splice/patch-coupling (SPC) pathway, the "sequential SPC" and the "SPC with fork collision", operate during DSB repair. In the 47 mutant with or without a das suppressor, coupling of the exchanges was greatly reduced, indicating a crucial role of the 47/46 complex in coupling of the genetic exchanges on the two sides of the DSB. From the observed dependence of the apparent coupling on the intracellular ratio of breakable and unbreakable chromosomes in different genetic backgrounds it is inferred that linking of the DNA ends by 47/46 protein is the mechanism that accounts for their concerted action during DSB repair. A mechanism of replicative resolution of D-loop intermediate (RR pathway) is suggested to explain the phenomenology of DSB repair in DNA arrest and uvsW mutants. A "left"-"right" bias in the recombinogenic action of two DNA ends of the broken chromosome was observed which was particularly prominent in the 59 (41-helicase loader) and 39 (topoisomerase) mutants. Phage topoisomerase II (gp39-52-60) is indispensable for growth in the DNA arrest mutants: the doubles 47(-)39(-), uvsX 39(-) and 59(-)39(-) are lethal.

Bacteriophage T4↗

PubMed2004

Elevated presence of retrotransposons at sites of DNA double strand break repair in mouse models of metabolic oxidative stress and MYC-induced lymphoma.

The chromosomally integrated shuttle vector pUR288 contains a lacZ reporter gene to study mutagenesis in vivo. We used pUR288 to compare patterns of genomic instability in two mouse models, lymphoma resulting from deregulated c-MYC expression (lambda-MYC), and endogenous oxidative stress caused by partial glucose 6-phosphate dehydrogenase (G6PD) deficiency. We found previously that spontaneous mutations in both models were predominantly genomic rearrangements of lacZ with mouse sequences, while most mutations in controls were point mutations. Here, we characterized the fine structure of 68 lacZ/mouse rearrangements from lambda-MYC lymphomas and G6PD deficient mice by sequencing breakpoint junctions and determining the origin of recombining mouse sequences. Fifty-eight of 68 (85%) recombination partners were identified. The structure of rearrangements from both lambda-MYC and G6PD deficient mice were remarkably alike. Intra-chromosomal deletions and inversions were common, occurring in 41% (24/58) of rearrangements, while 59% (34/58) were random translocations between lacZ and other chromosomes. Signatures of double strand break repair by nonhomologous end joining were observed at breakpoint junctions; 37% (25/68) contained 1-4 bp microhomologies, while the remaining breakpoints had no sequence homology. Long interspersed nuclear element-1 (LINE-1 or L1) retrotransposons, which constitute approximately 10% of the mouse genome, were present at 25% (17/68) of breakpoints, suggesting their participation in rearrangements. The similarity in the structure of rearrangements is consistent with the hypothesis that genetic rearrangements in lambda-MYC lymphomas and G6PD deficient mice result from the same mechanism, mutagenic repair of DNA double strand breaks arising from oxidative damage.

Animals↗

PubMed1995

What to do at an end: DNA double-strand-break repair.

Repairing chromosome breaks is essential to cell survival. A major lethal effect of ionizing radiation (IR) damage is the creation of double-strand DNA breaks. Recently, a number of mammalian cell mutants that are sensitive to IR damage have been described, revealing a unique repair pathway. The DNA-dependent protein kinase (DNA-PK) is necessary for double-strand-break repair and lymphoid V(D)J recombination. DNA-PK consists of three subunits: the Ku autoantigen heterodimer and a kinase (DNA-PKCS) that is deficient in mouse scid mutant cells.

Animals↗

PubMed2000

Reaction rate coefficients of hydroxyl radical-induced DNA single- and alpha-type double-strand breaks.

With a model system of pBR322 plasmid DNA solution in vitro, the dose effects of radiation-induced single- and double-strand breaks (SSB and DSB) were measured and DSB was distinguished into alpha- and beta-types. Under the condition of low scavenging capacity existing in the irradiated DNA solution, SSB and alpha DSB were mainly induced by hydroxyl radicals (.OH). Moreover, a certain relationship was obtained between the SSB and alpha DSB yields and the DNA concentration. It was found that when the DNA solution was irradiated in the presence of 2.5 mmol dm-3 mannitol, the reciprocals of G(SSB) and G(alpha DSB), respectively, were linearly related to the reciprocal of the DNA concentration, i.e. the competition reactions of DNA and mannitol for .OH radicals can be described by second-order kinetics. The rate coefficients and the efficiencies of the .OH radical inducing SSB were deduced. Also, the reaction rate coefficients and the efficiencies for the induction of alpha DSB from SSB by the .OH radical transfer mechanism, were first derived from the competition kinetics.

DNA↗

PubMed1990

Neutral filter elution detects only limited inhibition of double-strand break repair by 9-beta-D-arabinofuranosyladenine.

Bradley and Kohn (1979) showed that the neutral filter elution technique detects DNA double-strand breaks (dsb), yet there is still some uncertainty regarding the interpretation of results obtained with this technique (viz. the curvilinear dose-response curve and the rapid repair kinetics). In this report we have investigated the effect of the nucleoside analogue 9-beta-D-arabinofuranosyladenine (ara A), a known inhibitor of dsb repair, on the DNA repair in X-irradiated Ehrlich ascites tumour cells as measured by this technique. We have compared the effect of ara A on repair in these cells with results previously obtained with the same cell line and using the neutral velocity sedimentation and DNA unwinding techniques (Blöcher, 1982; Bryant and Blöcher, 1982). Our results suggest that the lesions measured by the neutral elution technique are different from those measured by neutral velocity sedimentation or long-term repair measured by DNA unwinding.

Animals↗

PubMed1997

Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family.

Meiotic recombination in S. cerevisiae is initiated by double-strand breaks (DSBs). In certain mutants, breaks accumulate with a covalently attached protein, suggesting that cleavage is catalyzed by the DSB-associated protein via a topoisomerase-like transesterase mechanism. We have purified these protein-DNA complexes and identified the protein as Spo11, one of several proteins required for DSB formation. These findings strongly implicate Spo11 as the catalytic subunit of the meiotic DNA cleavage activity. This is the first identification of a biochemical function for any of the gene products involved in DSB formation. Spo11 defines a protein family with other members in fission yeast, nematodes, and archaebacteria. The S. pombe homolog, rec12p, is also known to be required for meiotic recombination. Thus, these findings provide direct evidence that the mechanism of meiotic recombination initiation is evolutionarily conserved.

Amino Acid Sequence↗

PubMed1999

Discrimination between genotoxicity and cytotoxicity for the induction of DNA double-strand breaks in cells treated with aldehydes and diepoxides.

The time-dependent dose-response relationships for the induction of DNA double-strand breaks (DSB) assessed by pulsed-field gel electrophoresis (PFGE) and for viability (evaluated by the MTT cytotoxicity test) were investigated in order to discriminate between genotoxic and cytotoxic mechanisms of DNA fragmentation. Cultured human lung epithelial cells (A549) were treated (i) with the aldehydes formaldehyde or glutaraldehyde and (ii) with the DNA-DNA interstrand crosslinkers melphalan, diepoxybutane or diepoxyoctane. Induction of DSB by formaldehyde and glutaraldehyde was seen only after cell viability was reduced to less than about 60% of the control values, indicating that DSB were the consequence of extragenomic damage and viability loss. Melphalan, diepoxybutane and diepoxyoctane induced DSB by a genotoxic mode with concentrations that did not affect cell survival: 8 h after treatment initiation both heat-labile crosslinks and DSB could be detected. Cells were not able to repair the crosslinks induced by diepoxybutane, the crosslinker with the shortest chain length. In contrast, with melphalan and diepoxyoctane, which have a longer crosslinking property considerable repair of crosslinks was observed. The molecular size distribution of the produced DNA fragments supported this mechanistic distinction. The DNA fragments generated by diepoxides were initially large, their concentration decreasing monotonously from 7 Mbp to less than 1 Mbp and were converted to smaller fragments by 72 h in the course of cell death. In contrast, DNA fragments induced by formaldehyde peaked below 1 Mbp, implicating activation of DNA-degrading enzymes.

Aldehydes↗