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PubMed1999

Double-strand break repair in yeast requires both leading and lagging strand DNA polymerases.

Mitotic double-strand break (DSB)-induced gene conversion at MAT in Saccharomyces cerevisiae was analyzed molecularly in mutant strains thermosensitive for essential replication factors. The processivity cofactors PCNA and RFC are essential even to synthesize as little as 30 nucleotides following strand invasion. Both PCNA-associated DNA polymerases delta and epsilon are important for gene conversion, though a temperature-sensitive Pol epsilon mutant is more severe than one in Pol delta. Surprisingly, mutants of lagging strand replication, DNA polymerase alpha (pol1-17), DNA primase (pri2-1), and Rad27p (rad27 delta) also greatly inhibit completion of DSB repair, even in G1-arrested cells. We propose a novel model for DSB-induced gene conversion in which a strand invasion creates a modified replication fork, involving leading and lagging strand synthesis from the donor template. Replication is terminated by capture of the second end of the DSB.

Alleles↗

PubMed2004

Double-strand break of giant DNA: protection by glucosyl-hesperidin as evidenced through direct observation on individual DNA molecules.

We have performed a real-time observation of photo-induced breakage on individual long DNA molecules stained by a cyanine dye, YOYO, under the intense illumination (lambda=450-490 nm) in solution. It was shown that the double-strand breakage is accelerated by Fenton's reagent (Fe2+/H2O2). In addition, it was found that the breakage reaction is protected in the presence of a water-soluble flavonoid, glucosyl-hesperidin. To interpret the kinetic process of the double-strand breakage reaction, a two-step mechanism is proposed: under light illumination on giant DNA molecules, single-strand breaks are generated to induce nicks in a stochastic manner, and then reactive oxygen attacks these nicks causing the double-strand break.

Bacteriophage T4↗

PubMed1994

Combination of static-field gel electrophoresis and densitometric scanning for the determination of radiation-induced DNA double-strand breaks in CHO cells.

An experimental setup using static-field gel electrophoresis (SFGE) was developed to determine radiation-induced DNA double-strand breaks (DSBs) in CHO-K1 cells after exposure to X-rays or heavy charged particles. The fraction of DNA eluted into the gel matrix depends on the quantity of DSBs introduced. In agreement with a recent report, SFGE and pulsed-field electrophoresis were found to be equally sensitive in DSB detection. With radiolabeled DNA from cell cultures, the absolute amount of DNA migrating out of agarose plugs into the gel was quantified by determining the radioactivity in the gel lane. Alternatively, relative measurements of the amount of DNA released into the gel were achieved with a standardized protocol for both SFGE and a subsequent densitometric scanning of photographic negatives from gels stained with ethidium bromide. After calibration with the radioactive method, the fractions of DNA retained could be calculated directly from the data obtained with the densitometric assay to set up classical dose-effect curves. This procedure was validated for its application with heavy ions using an 500 MeV/u lead beam.

Animals↗

PubMed1999

Formation of single- and double-strand breaks of pBR322 plasmid irradiated in the presence of scavengers.

By the method of gel electrophoresis, radiation-induced DNA single- and double-strand breaks (SSB, DSB) were studied with a model system of pBR322 solution in vitro in the presence of 'OH radical scavengers, mannitol and TE (10(-2) mol dm(-3) Tris and 10(-3) mol dm(-3) ethylene diamine tetra-acetic acid). Experiments showed that SSB resulted from one-hit events of radiation energy deposition and DSB resulted from both one-hit and two-hit energy deposition events and so were distinguished into two classes of alphaDSB and betaDSB. Moreover, alpha/beta, where alpha is the number of DSB per unit dose induced in one irradiation event and beta the number of DSB per unit squared dose induced by the combination of two independent SSB, was related to the scavenging capacity, sigma, and for sigma > 10(8) s(-1), alphaDSB predominate over DSB. On the other hand, if sigma<2x10(8)s(-1), the measured G(alphaDSB) decreased in parallel with G(SSB), i.e., G(alphaDSB)/G(SSB) was a constant. When sigma>2x10(8) s(-1). G(alphaDSB) decreased slightly so that the ratio of alphaDSB to SSB evidently increased. Therefore, alphaDSB could be induced by the radical transfer mechanism for sigma<2x10(8) s(-1) and contrarily produced by the local multiply damaged sites (LMDS) mechanism for sigma larger than this value. In addition, the distance for two independent complementary SSB forming a DSB was deduced, but no apparent variation of it was found in the wide sigma range from approximately 10(5) to approximately 10(9) s(-)1, which shows that the DNA steric structure was not influenced by mannitol.

DNA Damage↗

PubMed2006

Polymorphisms in DNA double-strand break repair genes and risk of breast cancer: two population-based studies in USA and Poland, and meta-analyses.

The double-strand break DNA repair pathway has been implicated in breast carcinogenesis. We evaluated the association between 19 polymorphisms in seven genes in this pathway (XRCC2, XRCC3, BRCA2, ZNF350, BRIP1, XRCC4, LIG4) and breast cancer risk in two population-based studies in USA (3,368 cases and 2,880 controls) and Poland (1,995 cases and 2,296 controls). These data suggested weak associations with breast cancer risk for XRCC3 T241M and IVS7-14A>G (pooled odds ratio (95% confidence interval): 1.18 (1.04-1.34) and 0.85 (0.73-0.98) for homozygous variant vs wild-type genotypes, respectively), and for an uncommon variant in ZNF350 S472P (1.24 (1.05-1.48)), with no evidence for study heterogeneity. The remaining variants examined had no significant relationships to breast cancer risk. Meta-analyses of studies in Caucasian populations, including ours, provided some support for a weak association for homozygous variants for XRCC3 T241M (1.16 (1.04-1.30); total of 10,979 cases and 10,423 controls) and BRCA2 N372H (1.13 (1.10-1.28); total of 13,032 cases and 13,314 controls), and no support for XRCC2 R188H (1.06 (0.59-1.91); total of 8,394 cases and 8,404 controls). In conclusion, the genetic variants evaluated are unlikely to have a substantial overall association with breast cancer risk; however, weak associations are possible for XRCC3 (T241M and IVS7-14A>G), BRCA2 N372H, and ZNF350 S472P. Evaluation of potential underlying gene-gene interactions or associations in population subgroups will require even larger sample sizes.

Adult↗

PubMed1989

Repair of bleomycin-induced DNA double-strand breaks in Vicia faba.

As detected by neutral DNA elution, bleomycin induced at the concentrations tested (5, 10 and 50 micrograms/ml) DNA double-strand breaks (dsbs) in in vitro cultured embryos of V. faba. Most of these breaks were repaired during a 4-h incubation period after treatment. Dsbs also occurred after treatment with 2.5 and 5 mM of N-methyl-N-nitrosourea (MNU) but in contrast to those induced by bleomycin, these dsbs remained unrepaired during the 4-h incubation period following the treatment.

Bleomycin↗

PubMed1989

Induction of chromosomal aberrations in CHO cells by restriction endonucleases: effects of blunt- and cohesive-ended double-strand breaks in cells treated by 'pellet' methods.

In recent reports it has been suggested that restriction endonucleases (RE) producing cohesive-ended double-strand breaks (dsb), are of comparable effectiveness to those producing blunt-ended dsb in causing chromosomal aberrations (CA) in mammalian cells. In several of these reports, trypsinized cells or suspension cultures were treated as cell 'pellets' in small volumes containing RE and storage buffers. In this study we have examined this by comparing 2 'pellet' methods in which trypsinized Chinese hamster cells were treated with RE in small volumes, after cells were centrifuged to a pellet. In the first method, cells were treated with RE in storage buffer as previously reported (e.g. Obe et al., 1985). In the second method, cells were treated as pellets with Sendai virus and purified RE. For both methods we show that the frequency of chromosomal aberrations was higher in cells treated with RE causing blunt-ended dsb than those causing cohesive-ended dsb. The first method however was found to lead to substantial loss in cell viability. The results strengthen the conclusion drawn from our earlier work, using treatment of attached V79 or CHO-K1 cells with Sendai virus, that cohesive-ended dsb are less effective than blunt-ended dsb in causing chromosomal aberrations.

Animals↗

PubMed1995

Dose-rate effect on induction and repair rate of radiation-induced DNA double-strand breaks in a normal and an ataxia telangiectasia human fibroblast cell line.

Using pulsed-field gel electrophoresis (PGFE), we measured DNA double-strand breaks (DSB) in a normal human fibroblast and in a cell line derived from a patient suffering from ataxia telangiectasia (AT), a syndrome associated with a hypersensitivity to ionizing radiation. Initial DSB levels assessed after irradiation at 4 degrees C are similar in both cell lines. The DSB repair rate was measured after 30 Gy delivered at 4 degrees C and followed by an incubation at 37 degrees C for 24 h. In AT cells, the DSB repair rate is faster between 0.5 and 9 h and slower between 9 and 24 h. In addition, the DSB levels were measured after irradiation at 37 degrees C at 0.01 Gy min-1 (5-40 Gy). The shape of the curves was curvilinear and a plateau was reached at 10 Gy in the control. After an irradiation at 37 degrees C, DSB levels were significantly higher in AT cells than in the normal fibroblast cells. A model was developed assuming that DSB induction is independent of temperature and that DSB repair rate is independent of dose-rate and dose. This model was used to predict the 37 degrees C DSB data on the basis of the 4 degrees C data. Experimental data and predictions are in agreement, thus validating the above assumptions. It is suggested that, even for extreme situations such as 30 Gy delivered at 4 degrees C or 30 Gy delivered at 37 degrees C at 0.01 Gy min-1, DSB induction and repair are identical. Our results could be interpreted assuming an heterogeneity of DSB. A small fraction of DSB is slowly repaired. This fraction is lower in control than in AT cells. By protracting repair time, the 37 degrees C low-dose rate experiments permit a cleaner distinction between AT and control cells.

Ataxia Telangiectasia↗

PubMed1991

Differences in accumulation of blunt- and cohesive-ended double-strand breaks generated by restriction endonucleases in electroporated CHO cells.

Restriction endonucleases (RE) have been used in cytogenetic studies to mimic the DNA double-strand break (dsb)-inducing action of radiation. In the experiments presented here, we have treated electroporated CHO cells with RE and have measured the resulting dsb using the filter elution technique under non-denaturing conditions (pH 9.6). PvuII, which generates blunt-ended dsb, gave rise to a significant number of measurable dsb. The frequency of the dsb induced by PvuII is shown to increase over a 3-12-h post-treatment incubation period, which implies that the RE is active in the cell for a considerable length of time. We postulate that the accumulation of dsb reflects a competition between enzymatic incision and repair of the DNA. The presence of araA, a known inhibitor of DNA synthesis, did not affect the frequency of PvuII-induced breaks indicating a lack of an inhibitory effect of araA on the repair of RE-induced dsb. Two RE which cause cohesive-ended dsb, namely BamHI and EcoRI, were found to be ineffective in giving rise to measurable dsb. Our interpretation of this is that for cohesive-ended dsb (caused by BamHI and EcoRI) the rate at which these breaks are rejoined matches or exceeds the rate of enzymatic incision and hence no dsb were observed. In the case of PvuII, the possibly slower rate of repair of blunt-ended termini would on this hypothesis result in the observed net accumulation of dsb.

Animals↗

PubMed2006

Epidermal growth factor receptor and DNA double strand break repair: the cell's self-defence.

The purpose of this review is to discuss the relation between the repair of DNA double strand breaks (DSB)--the main lethal lesion inflicted by ionising radiation-and the function of receptors of epidermal growth factor (EGFR) and similar ligands (other members of the ERBB family). The reviewed experimental data support the assumption that in mammalian cells, one consequence of EGFR/ERBB activation by X-rays is its internalisation and nuclear translocation together with DNA-dependent protein kinase (DNA-PK) subunits present in lipid rafts or cytoplasm. The effect of EGFR/ERBB stimulation on DSB rejoining would be due to an increase in the nuclear content of DNA-PK subunits and hence, in activity increase of the DNA-PK dependent non-homologous end-joining (D-NHEJ) system. Such mechanism explains the radiosensitising action of "membrane-active drugs", hypertonic media, and other agents that affect nuclear translocation of proteins. Also, one radiosensitising effect of the recently introduced into clinical practice EGFR/ERBB inhibitors would consist on counteracting the nuclear translocation of DNA-PK subunits. In result, D-NHEJ may be less active in inhibitor-treated cells and this will contribute to an enhanced lethal effect of irradiation. The reviewed observations point to a heretofore not understood mechanism of the cell's self-defence against X-rays which can be exploited in combined radio- and chemotherapy.

Animals↗

PubMed2004

RAG proteins shepherd double-strand breaks to a specific pathway, suppressing error-prone repair, but RAG nicking initiates homologous recombination.

The two major pathways for repairing double-strand breaks (DSBs), homologous recombination and nonhomologous end joining (NHEJ), have traditionally been thought to operate in different stages of the cell cycle. This division of labor is not absolute, however, and precisely what governs the choice of pathway to repair a given DSB has remained enigmatic. We pursued this question by studying the site-specific DSBs created during V(D)J recombination, which relies on classical NHEJ to repair the broken ends. We show that mutations that form unstable RAG postcleavage complexes allow DNA ends to participate in both homologous recombination and the error-prone alternative NHEJ pathway. By abrogating a key function of the complex, these mutations reveal it to be a molecular shepherd that guides DSBs to the proper pathway. We also find that RAG-mediated nicks efficiently stimulate homologous recombination and discuss the implications of these findings for oncogenic chromosomal rearrangements, evolution, and gene targeting.

Animals↗

PubMed2000

Relationship between DNA double-strand breaks, cell killing, and fibrosis studied in confluent skin fibroblasts derived from breast cancer patients.

PURPOSE: To investigate the relationship between DNA double-strand breaks (dsbs), cell killing, and fibrosis using skin fibroblasts derived from breast cancer patients who received postmastectomy radiotherapy. METHODS AND MATERIALS: Experiments were performed with 12 lines of normal skin fibroblasts derived from recurrence-free breast cancer patients. Cells were irradiated in confluence and cell survival was determined either after immediate or delayed (14 h) plating using a colony-forming assay. Dsbs were measured by constant-field gel electrophoresis. The "excess risk of fibrosis" was previously scored by Johansen et al. (IJRB 1994;66:407-412). RESULTS: The 12 cell lines showed a typical spectrum of radiosensitivity. The mean value of surviving fraction after 3.5 Gy (SF3.5) was 0.063 for immediate and 0.174 for delayed plating with a coefficient of variation (CV) of 44 and 39%, respectively. There was also a broad variation in the extent of recovery from potentially lethal damage (RPLD), which was not correlated with the immediate sensitivity. The number of initial dsbs as well as the half-times of dsb repair showed little variation, whereas there were considerable differences in the number of residual dsbs (CV = 29%). The number of residual dsbs after 100 Gy was correlated significantly only with SF3.5 after delayed (r2 = O.59; p = 0.006) but not after immediate plating (r2 = 0.21, p = 0.16). There was also no significant relationship between residual dsbs and the "excess risk of fibrosis" determined for the respective patients. CONCLUSION: It is shown that the number of residual dsbs measured in confluent human fibroblast lines can be used to predict the cellular radiosensitivity after delayed but not after immediate plating and also not to predict the excess risk of fibrosis of the respective breast cancer patients.

Breast Neoplasms↗

PubMed1998

Telomeres and double-strand breaks: trying to make ends meet.

Eukaryotic cells encounter two types of DNA ends: telomeres, the natural ends of linear chromosomes, and double-strand breaks, resulting from DNA damage or normal chromosomal processes such as meiotic or V(D)J recombination. These two termini have long been seen as functionally distinct, based on whether they are resistant to fusion with other ends or instead are acted upon by the DNA-repair machinery. However, a series of recent papers has shown that members of a set of proteins that are crucial for the rejoining of DNA strand breaks are also required for normal telomere function, raising new questions about how these two types of termini maintain their functional distinction.

Antigens, Nuclear↗

PubMed2003

Different pathways of homologous recombination are used for the repair of double-strand breaks within tandemly arranged sequences in the plant genome.

Different DNA repair pathways that use homologous sequences in close proximity to genomic double-strand breaks (DSBs) result in either an internal deletion or a gene conversion. We determined the efficiency of these pathways in somatic plant cells of transgenic Arabidopsis lines by monitoring the restoration of the beta-glucuronidase (GUS) marker gene. The transgenes contain a recognition site for the restriction endonuclease I-SceI either between direct GUS repeats to detect deletion formation (DGU.US), or within the GUS gene to detect gene conversion using a nearby donor sequence in direct or inverted orientation (DU.GUS and IU.GUS). Without expression of I-SceI, the frequency of homologous recombination (HR) was low and similar for all three constructs. By crossing the different lines with an I-SceI expressing line, DSB repair was induced, and resulted in one to two orders of magnitude higher recombination frequency. The frequencies obtained with the DGU.US construct were about five times higher than those obtained with DU.GUS and IU.GUS, irrespective of the orientation of the donor sequence. Our results indicate that recombination associated with deletions is the most efficient pathway of homologous DSB repair in plants. However, DSB-induced gene conversion seems to be frequent enough to play a significant role in the evolution of tandemly arranged gene families like resistance genes.

Arabidopsis↗

PubMed2000

Oxidation of cellular thiols by hydroxyethyldisulphide inhibits DNA double-strand-break rejoining in G6PD deficient mammalian cells.

PURPOSE: We investigated the effect of protein- and non protein-thiol oxidation on DNA double-strand-break (DSB) rejoining after irradiation and its relevance in the survival of CHO cells. MATERIALS AND METHODS: We used mutant cells null for glucose 6 phosphate dehydrogenase (G6PD) activity since reducing equivalents, required for reduction of oxidized thiols, are typically generated through G6PD regulated production of NADPH. Cellular thiols were oxidized by pre-incubating the cells with hydroxyethyldisulphide (HEDS), the oxidized form of mercaptoethanol (ME). The concentrations of the intracellular and extracellular non-protein thiols (NPSH), glutathione, cysteine and mercaptoethanol were quantitated by HPLC. Protein thiols (PSH) were estimated using Ellman's reagent. Cell survival was determined by clonogenic assay. The induction and rejoining of DSB in cells was quantitated by Pulse Field Gel Electrophoresis after exposure to ionizing radiation. RESULTS: Much lower bioreduction of HEDS was found in the G6PD deficient mutants (E89) than in the wild-type cells (K1). A 1 h treatment of E89 cells with HEDS produced almost complete depletion of non-protein thiol (NPSH) and a 26% decrease in protein thiols. Only minor changes were found under similar conditions with K1 cells. When exposed to gamma radiation in the presence of HEDS, the G6PD null mutants exhibited a higher cell killing and decreased rate and extent of rejoining of DSB than were observed in K1 cells. Moreover, when the G6PD deficient cells were transfected with the gene encoding wild-type G6PD (A1A), they recovered close to wild-type cellular thiol status, cell survival and DSB rejoining. CONCLUSIONS: These results suggest that a functioning oxidative pentose phosphate pathway is required for DSB rejoining in cells exposed to a mild thiol oxidant.

Animals↗

PubMed1997

Hypersensitivity of ataxia telangiectasia fibroblasts to ionizing radiation is associated with a repair deficiency of DNA double-strand breaks.

We have studied the intrinsic radiosensitivity, repair of potentially lethal damage (PLD) and the repair rate of radiation-induced DNA double-strand breaks (DSB) in 11 non-transformed human fibroblast cell lines, four of which were homozygous for the A-T mutation and two that were heterozygous (A-TH). All the experiments were done on cells in plateau phase of growth (97-99% of cells in G0/G1). With a dose of 30 Gy delivered at 4 degrees C, the A-T cell lines had faster repair rates of up to 6 h, after which the repair curve crossed that of the control so that the residual damage at 24 h was higher in the A-T cells. Irradiation at 37 degrees C at low dose rate 1 cGy.min-1) produced even more marked differences between the A-T cells and controls: the residual DSB level was always higher in A-T cells than controls at doses of 5-40 Gy, due to defective repair of a small fraction of DSB in A-T cells. The two protocols showed DSB repair rates for the A-TH cell lines that were intermediate between those of the A-T and control cells. There was a quantitative relationship between the residual DSB after irradiation at 37 degrees C and the intrinsic radiosensitivity, and with the extent of PLD repair. There were very few apoptotic cells in the non-transformed control and A-T cell line, both before and after irradiation. In combination, these result support the contention that the defective repair of DSB is a mechanism of the hypersensitivity linked to the A-T mutation.

Ataxia Telangiectasia↗

PubMed2006

Polymorphisms in genes involved in DNA double-strand break repair pathway and susceptibility to benzene-induced hematotoxicity.

Benzene is a recognized hematotoxicant and carcinogen that produces genotoxic damage. DNA double-strand breaks (DSB) are one of the most severe DNA lesions caused directly and indirectly by benzene metabolites. DSB may lead to chromosome aberrations, apoptosis and hematopoietic progenitor cell suppression. We hypothesized that genetic polymorphisms in genes involved in DNA DSB repair may modify benzene-induced hematotoxicity. We analyzed one or more single nucleotide polymorphisms (SNPs) in each of seven candidate genes (WRN, TP53, NBS1, BRCA1, BRCA2, XRCC3 and XRCC4) in a study of 250 workers exposed to benzene and 140 controls in China. Four SNPs in WRN (Ex4 -16 G > A, Ex6 +9 C > T, Ex20 -88 G > T and Ex26 -12 T > G), one SNP in TP53 (Ex4 +119 C > G) and one SNP in BRCA2 (Ex11 +1487 A > G) were associated with a statistically significant decrease in total white blood cell (WBC) counts among exposed workers. The SNPs in WRN and TP53 remained significant after accounting for multiple comparisons. One or more SNPs in WRN had broad effects on WBC subtypes, with significantly decreased granulocyte, total lymphocyte, CD4(+)-T cell, CD8(+)-T cell and monocyte counts. Haplotypes of WRN were associated with decreased WBC counts among benzene-exposed subjects. Likewise, subjects with TP53 Ex4 +119 C > G variant had reduced granulocyte, CD4(+)-T cell and B cell counts. The effect of BRCA2 Ex11 +1487 A > G polymorphism was limited to granulocytes. These results suggest that genetic polymorphisms in WRN, TP53 and BRCA2 that maintain genomic stability impact benzene-induced hematotoxicity.

Adult↗

PubMed2004

Two modes of DNA double-strand break repair are reciprocally regulated through the fission yeast cell cycle.

Several considerations suggest that levels of the two major modes of double-strand break (DSB) repair, homologous recombination (HR), and nonhomologous end joining (NHEJ), are regulated through the cell cycle. However, this idea has not been explicitly tested. In the absence of the telomere-binding protein Taz1, fission yeast undergo lethal telomere fusions via NHEJ. These fusions occur only during periods of nitrogen starvation and fail to accumulate during logarithmic growth, when the majority of cells are in G2. We show that G1 arrest is the specific nitrogen starvation-induced event that promotes NHEJ between taz1(-) telomeres. Furthermore, the general levels of NHEJ and HR are reciprocally regulated through the cell cycle, so that NHEJ is 10-fold higher in early G1 than in other cell cycle stages; the reverse is true for HR. Whereas NHEJ is known to be dispensable for survival of DSBs in cycling cells, we find that it is critical for repair and survival of DSBs arising during G1.

Cell Cycle↗