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PubMed1999

I-SceI endonuclease, a new tool for studying DNA double-strand break repair mechanisms in Drosophila.

As a step toward the development of a homologous recombination system in Drosophila, we have developed a methodology to target double-strand breaks (DSBs) to a specific position in the Drosophila genome. This method uses the mitochondrial endonuclease I-SceI that recognizes and cuts an 18-bp restriction site. We find that >6% of the progeny derived from males that carry a marker gene bordered by two I-SceI sites and that express I-SceI in their germ line lose the marker gene. Southern blot analysis and sequencing of the regions surrounding the I-SceI sites revealed that in the majority of the cases, the introduction of DSBs at the I-SceI sites resulted in the complete deletion of the marker gene; the other events were associated with partial deletion of the marker gene. We discuss a number of applications for this novel technique, in particular its use to study DSB repair mechanisms.

Animals↗

PubMed2005

Biotinylation of K12 in histone H4 decreases in response to DNA double-strand breaks in human JAr choriocarcinoma cells.

We tested the hypothesis that biotinylation of K12 in histone H4 plays a role in the cellular response to double-strand breaks (DSB) of DNA in human cells. DSB were caused by treating choriocarcinoma JAr cells with etoposide. Biotinylation of K12 in histone H4 decreased by 50% as early as 10-20 min after initiation of treatment with etoposide. Biotinylation returned to initial levels 30-40 min after the addition of etoposide to the medium. Temporal patterns of K12-biotinylation were similar for human lymphoma cells. Phosphorylation of S14 of histone H2B and poly(ADP-ribosylation) of glutamate residues on histone H2A are known markers of DSB in DNA; these modifications increased 10-40 min after alterations in K12-biotinylation occurred. Decreased biotinylation of K12 of histone H4 was specific for DSB but was not detectable in response to single-strand breaks or the formation of thymine dimers. Biotin-deficient choriocarcinoma cells exhibited a 40% decrease in rates of survival in response to etoposide compared with biotin-sufficient controls. These studies suggest that the lack of biotinylation of K12 in histone H4 is an early signaling event in response to DSB.

Antineoplastic Agents, Phytogenic↗

PubMed1998

Double-strand break repair in Ku86- and XRCC4-deficient cells.

The Ku86 and XRCC4 proteins perform critical but poorly understood functions in the repair of DNA double-strand breaks. Both Ku 86- and XRCC4-deficient cells exhibit profound radiosensitivity and severe defects in V(D)J recombination, including excessive deletions at recombinant junctions. Previous workers have suggested that these phenomena may reflect defects in joining of the broken DNA ends or in protection of the ends from nucleases. However, end joining in XRCC4-deficient cells has not been examined. Here we show that joining of both matched and mismatched DNA ends occurs efficiently in XRCC4-deficient cells. Furthermore, analysis of junctions shows that XRCC4 is not required to protect the ends from degradation. However, nucleotide sequence analysis of junctions derived from joining of mismatched DNA ends in XRCC4-deficient cells revealed a strong preference for a junction containing a 7 nt homology. Similar results were obtained in Ku86-deficient cells. These data suggest that in the absence of XRCC4 or Ku86, joining is assisted by base pairing interactions, supporting the hypothesis that these proteins may participate in aligning or stabilizing intermediates in end joining.

Animals↗

PubMed1991

A unique pathway of double-strand break repair operates in tandemly repeated genes.

The RAD52 gene product of the yeast Saccharomyces cerevisiae is required for most spontaneous recombination and almost all double-strand break (DSB) repair. In contrast to recombination elsewhere in the genome, recombination in the ribosomal DNA (rDNA) array is RAD52 independent. To determine the fate of a DSB in the rDNA gene array, a cut site for the HO endonuclease was inserted into the rDNA in a strain containing an inducible HO gene. DSBs were efficiently repaired at this site, even in the absence of the RAD52 gene product. Efficient RAD52-independent DSB repair was also observed at another tandem gene array, CUP1, consisting of 18 repeat units. However, in a smaller CUP1 array, consisting of only three units, most DSBs (ca. 80%) were not repaired and resulted in cell death. All RAD52-independent DSB repair events examined resulted in the loss of one or more repeat units. We propose a model for DSB repair in repeated sequences involving the generation of single-stranded tails followed by reannealing.

Blotting, Southern↗

PubMed2001

Evidence for biased holliday junction cleavage and mismatch repair directed by junction cuts during double-strand-break repair in mammalian cells.

In mammalian cells, several features of the way homologous recombination occurs between transferred and chromosomal DNA are consistent with the double-strand-break repair (DSBR) model of recombination. In this study, we examined the segregation patterns of small palindrome markers, which frequently escape mismatch repair when encompassed within heteroduplex DNA formed in vivo during mammalian homologous recombination, to test predictions of the DSBR model, in particular as they relate to the mechanism of crossover resolution. According to the canonical DSBR model, crossover between the vector and chromosome results from cleavage of the joint molecule in two alternate sense modes. The two crossover modes lead to different predicted marker configurations in the recombinants, and assuming no bias in the mode of Holliday junction cleavage, the two types of recombinants are expected in equal frequency. However, we propose a revision to the canonical model, as our results suggest that the mode of crossover resolution is biased in favor of cutting the DNA strands upon which DNA synthesis is occurring during formation of the joint molecule. The bias in junction resolution permitted us to examine the potential consequences of mismatch repair acting on the DNA breaks generated by junction cutting. The combination of biased junction resolution with both early and late rounds of mismatch repair can explain the marker patterns in the recombinants.

Animals↗

PubMed2003

Pathways of DNA double-strand break repair during the mammalian cell cycle.

Little is known about the quantitative contributions of nonhomologous end joining (NHEJ) and homologous recombination (HR) to DNA double-strand break (DSB) repair in different cell cycle phases after physiologically relevant doses of ionizing radiation. Using immunofluorescence detection of gamma-H2AX nuclear foci as a novel approach for monitoring the repair of DSBs, we show here that NHEJ-defective hamster cells (CHO mutant V3 cells) have strongly reduced repair in all cell cycle phases after 1 Gy of irradiation. In contrast, HR-defective CHO irs1SF cells have a minor repair defect in G(1), greater impairment in S, and a substantial defect in late S/G(2). Furthermore, the radiosensitivity of irs1SF cells is slight in G(1) but dramatically higher in late S/G(2), while V3 cells show high sensitivity throughout the cell cycle. These findings show that NHEJ is important in all cell cycle phases, while HR is particularly important in late S/G(2), where both pathways contribute to repair and radioresistance. In contrast to DSBs produced by ionizing radiation, DSBs produced by the replication inhibitor aphidicolin are repaired entirely by HR. irs1SF, but not V3, cells show hypersensitivity to aphidicolin treatment. These data provide the first evaluation of the cell cycle-specific contributions of NHEJ and HR to the repair of radiation-induced versus replication-associated DSBs.

Animals↗

PubMed2006

Tying the loose ends together in DNA double strand break repair with 53BP1.

To maintain genomic stability and ensure the fidelity of chromosomal transmission, cells respond to various forms of genotoxic stress, including DNA double-stranded breaks (DSBs), through the activation of DNA damage response signaling networks. In response to DSBs as induced by ionizing radiation (IR), during DNA replication, or through immunoglobulin heavy chain (IgH) rearrangements in B cells of lymphoid origin, the phosphatidyl inositol-like kinase (PIK) kinases ATM (mutated in ataxia telangiectasia), ATR (ATM and Rad3-related kinase), and the DNA-dependent protein kinase (DNA-PK) activate signaling pathways that lead to DSB repair. DSBs are repaired by either of two major, non-mutually exclusive pathways: homologous recombination (HR) that utilizes an undamaged sister chromatid template (or homologous chromosome) and non- homologous end joining (NHEJ), an error prone mechanism that processes and joins broken DNA ends through the coordinated effort of a small set of ubiquitous factors (DNA-PKcs, Ku70, Ku80, artemis, Xrcc4/DNA lig IV, and XLF/Cernunnos). The PIK kinases phosphorylate a variety of effector substrates that propagate the DNA damage signal, ultimately resulting in various biological outputs that influence cell cycle arrest, transcription, DNA repair, and apoptosis. A variety of data has revealed a critical role for p53-binding protein 1 (53BP1) in the cellular response to DSBs including various aspects of p53 function. Importantly, 53BP1 plays a major role in suppressing translocations, particularly in B and T cells. This report will review past experiments and current knowledge regarding the role of 53BP1 in the DNA damage response.

Journal Article↗

PubMed1989

Radioprotection of cultured mammalian cells by the aminothiols WR-1065 and WR-255591: correlation between protection against DNA double-strand breaks and cell killing after gamma radiation.

We compared the effects of the radioprotective aminothiols WR-1065 and WR-255591 on the induction of DNA double-strand breaks (DSBs) and on the survival of aerated Chinese hamster ovary cells exposed to 60Co gamma radiation. DSBs were measured using the pH 9.6 neutral elution method. In agreement with earlier studies, protection factors for both drugs measured using the end point of clonogenic cell survival were significantly greater than the protection factors for DSB induction when DSBs were measured after gamma-ray doses ranging from 20 to 90 Gy. However, when DSBs and cell survival measurements were made on the same cell populations after low radiation doses (between 3 and 30 Gy) using the replicate plating method, there appeared to be a close correlation between the modification of DSB induction and the modification of cell survival produced by both drugs. The major influence accounting for the differences between these and previously obtained results appears to be the range of radiation doses used, suggesting that protection against DSB induction is radiation-dose dependent.

Animals↗

PubMed1995

Methods for the quantification of DNA double-strand breaks determined from the distribution of DNA fragment sizes measured by pulsed-field gel electrophoresis.

Different methods were used for evaluating data for DNA double-strand breaks (DSBs), as obtained by pulsed-field gel electrophoresis (PFGE) after X irradiation of Chinese hamster ovary cells. A total of 60 data points in the dose range of 0 to 116 Gy, along with repair data for 30 and 60 Gy, were analyzed by four methods: (1) percentage of DNA released from the plug, (2) specific size markers (percentage of DNA less than specific sizes, (3) fragment size distributions and (4) shape of the molecular weight (M) distributions. With the last method, both the slope and the intercept of the logarithm of the amount of radioactive DNA/delta M/M plotted as a function of M were used for calculating DSBs/100 Mbp. The slope and the intercept analyses differ in that the former is relatively independent of DNA trapped in the agarose plugs, i.e. cannot be released by doses of 100-150 Gy, whereas the intercept is dependent on the percentage of DNA trapped. Also, calculations of DSBs/100 Mbp for methods 1, 2 and 3 depend on the amount of DNA trapped in the plug. However, the slope method is unreliable for doses below about 20 Gy, and the scatter of data points is much greater than that obtained by the intercept method and by methods 1, 2 and 3. Therefore, the fragment size distribution and the specific size marker methods give the most consistent results, with 0.49 +/- 0.03 (95% CI) (DSBs/100 Mbp)/Gy. With the specific size marker method, however, care must be taken in selection of size markers in relation to the levels of DSBs of interest. Assuming randomly distributed DSBs, all four methods gave essentially the same results; i.e., the dose response was linear with a calculated level of 0.5-0.6 (DSBs/100 Mbp)/Gy, which is the same as 0.47-0.62 determined previously by calibrating with 125IdU.

Animals↗

PubMed1999

Wortmannin sensitizes mammalian cells to radiation by inhibiting the DNA-dependent protein kinase-mediated rejoining of double-strand breaks.

Wortmannin has been shown to be an efficient radiosensitizer. Since wortmannin is able to inhibit DNA-dependent protein kinase (DNA-PK) and double-strand break (DSB) rejoining, it is believed that its mechanism of radiation sensitization is through the inhibition of DNA-PK-mediated repair of DSBs. However, since wortmannin is not a specific inhibitor, the possibility that other kinases are inhibited and thereby may contribute to radiosensitization cannot be ruled out. Here we present data confirming the radiosensitizing effect of wortmannin on cells of different cell lines. In the same range of wortmannin concentrations, survival after exposure to ionizing radiation correlated well with DSB rejoining and the induction of micronuclei, suggesting that the inhibition of the processing of DSBs is involved in the sensitizing effect. Pretreatment with wortmannin enhanced the radiosensitivity of ataxia telangiectasia (AT) cells, thereby precluding the participation of ATM protein in the radiation sensitization by wortmannin. At the same time, irradiated DNA-PK-deficient cells were not significantly affected by pretreatment with wortmannin. These observations support a likely mechanism; that is, wortmannin sensitizes cells to radiation through inhibition of the DNA-PK-mediated rejoining of DSBs.

Androstadienes↗

PubMed2005

Carcinogenic lead chromate induces DNA double-strand breaks in human lung cells.

Hexavalent chromium (Cr(VI)) is a widespread environmental contaminant and a known human carcinogen, generally causing bronchial cancer. Recent studies have shown that the particulate forms of Cr(VI) are the potent carcinogens. Particulate Cr(VI) is known to induce a spectrum of DNA damage such as DNA single strand breaks, Cr-DNA adducts, DNA-protein crosslinks and chromosomal aberrations. However, particulate Cr(VI)-induced DNA double strand breaks (DSBs) have not been reported. Thus, the aim of this study was to determine if particulate Cr(VI)-induces DSBs in human bronchial cells. Using the single cell gel electrophoresis assay (comet assay), showed that lead chromate-induced concentration dependent increases in DSBs with 0.1, 0.5, 1 and 5 microg/cm2 lead chromate inducing a 20, 50, 67 and 109% relative increase in the tail integrated intensity ratio, respectively. Sodium chromate at concentrations of 1, 2.5 and 5 microM induced 38, 78 and 107% relative increase in the tail integrated intensity ratio, respectively. We also show that genotoxic concentrations of lead chromate activate the ataxia telangiectasia mutated (ATM) protein, which is thought to play a central role in the early stages of DSB detection and controls cellular responses to this damage. The H2A.X protein becomes rapidly phosphorylated on residue serine 139 in cells when DSBs are introduced into the DNA by ionizing radiation. By using immunofluorescence, we found that lead chromate-induced concentration-dependent increases in phosphorylated H2A.X (r-H2A.X) foci formation with 0.1, 0.5, 1, 5 and 10 microg/cm2 lead chromate inducing a relative increase in the number of cells with r-H2A.X foci formation of 43, 51, 115 and 129%, respectively.

Ataxia Telangiectasia Mutated Proteins↗

PubMed1984

Transposase promotes double strand breaks and single strand joints at Tn10 termini in vivo.

We present evidence that Tn10 transposase promotes double strand breaks and single strand joints at Tn10 termini in vivo. Plasmids containing a shortened Tn10 element and a transposase overproducer fusion give rise, upon transposase induction, to new DNA species. The most prominent class is a circularized transposon molecule whose structure suggests that it arises from double strand breakage at the two transposon ends followed by covalent joining between the 3' and 5' ends of one of the two strands. We have used formation of the circularized transposon as a physical assay for the interaction between transposase and different mutant and wild-type termini. These experiments show that transposase protein interacts preferentially with the genetically most active termini in a way that suppresses productive interaction with weaker termini present on the same substrate molecule.

Base Sequence↗

PubMed2006

The role of DNA double-strand breaks in spontaneous homologous recombination in S. cerevisiae.

Homologous recombination (HR) is a source of genomic instability and the loss of heterozygosity in mitotic cells. Since these events pose a severe health risk, it is important to understand the molecular events that cause spontaneous HR. In eukaryotes, high levels of HR are a normal feature of meiosis and result from the induction of a large number of DNA double-strand breaks (DSBs). By analogy, it is generally believed that the rare spontaneous mitotic HR events are due to repair of DNA DSBs that accidentally occur during mitotic growth. Here we provide the first direct evidence that most spontaneous mitotic HR in Saccharomyces cerevisiae is initiated by DNA lesions other than DSBs. Specifically, we describe a class of rad52 mutants that are fully proficient in inter- and intra-chromosomal mitotic HR, yet at the same time fail to repair DNA DSBs. The conclusions are drawn from genetic analyses, evaluation of the consequences of DSB repair failure at the DNA level, and examination of the cellular re-localization of Rad51 and mutant Rad52 proteins after introduction of specific DSBs. In further support of our conclusions, we show that, as in wild-type strains, UV-irradiation induces HR in these rad52 mutants, supporting the view that DNA nicks and single-stranded gaps, rather than DSBs, are major sources of spontaneous HR in mitotic yeast cells.

Alleles↗

PubMed2006

Modeling oncogenic translocations: distinct roles for double-strand break repair pathways in translocation formation in mammalian cells.

Reciprocal chromosomal translocations are implicated in the etiology of many tumors, including leukemias, lymphomas, and sarcomas. DNA double-strand breaks (DSBs) caused by various cellular processes and exogenous agents are thought to be responsible for the generation of most translocations. Mammalian cells have multiple pathways for repairing DSBs in the chromosomes: non-homologous end-joining (NHEJ), homologous recombination (HR), and single-strand annealing (SSA), which is a specialized pathway involving sequence repeats. In this review, we summarize the various reporters that have been used to examine the potential for each of these DSB repair pathways to mediate translocation formation in mammalian cells. This approach has demonstrated that NHEJ is very proficient at mediating translocation formation, while HR is not because of crossover suppression. Although SSA can efficiently mediate translocations between identical repeats, its contribution to translocation formation is likely very limited because of sequence divergence between repetitive elements in the genome.

Alu Elements↗

PubMedDate not supplied

Double strand break rejoining by the Ku-dependent mechanism of non-homologous end-joining.

The DNA-dependent protein kinase functions in the repair of DNA double strand breaks (DSBs) and in V(D)J recombination. To gain insight into the function of DNA-PK in this process we have carried out a mutation analysis of Ku80 and DNA-PKcs. Mutations at multiple sites within the N-terminal two thirds of Ku80 result in loss of Ku70/80 interaction, loss of DNA end-binding activity and inability to complement Ku80 defective cell lines. In contrast, mutations in the carboxy terminal region of the protein do not impair DNA end-binding activity but decrease the ability of Ku to activate DNA-PK. To gain insight into important functional domains within DNA-PKcs, we have analysed defective mutants, including the mouse scid cell line, and the rodent mutants, irs-20 and V-3. Mutational changes in the carboxy terminal region have been identified in all cases. Our results strongly suggest that the C-terminus of DNA-PKcs is required for kinase activity.

Animals↗

PubMed2002

Repair of sequence-specific 125I-induced double-strand breaks by nonhomologous DNA end joining in mammalian cell-free extracts.

In mammalian cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of double-strand break (DSB) repair. Rejoining of DSB produced by decay of (125)I positioned against a specific target site in plasmid DNA via a triplex-forming oligonucleotide (TFO) was investigated in cell-free extracts from Chinese hamster ovary cells. The efficiency and quality of NHEJ of the "complex" DSB induced by the (125)I-TFO was compared with that of "simple" DSB induced by restriction enzymes. We demonstrate that the extracts are indeed able to rejoin (125)I-TFO-induced DSB, although at approximately 10-fold decreased efficiency compared with restriction enzyme-induced DSB. The resulting spectrum of junctions is highly heterogeneous exhibiting deletions (1-30 bp), base pair substitutions, and insertions and reflects the heterogeneity of DSB induced by the (125)I-TFO within its target site. We show that NHEJ of (125)I-TFO-induced DSB is not a random process that solely depends on the position of the DSB but is driven by the availability of microhomology patches in the target sequence. The similarity of the junctions obtained with the ones found in vivo after (125)I-TFO-mediated radiodamage indicates that our in vitro system may be a useful tool to elucidate the mechanisms of ionizing radiation-induced mutagenesis and repair.

Animals↗

PubMed2001

Overexpression of the DNA-binding domain of poly(ADP-ribose) polymerase inhibits rejoining of ionizing radiation-induced DNA double-strand breaks.

PURPOSE: To assess the influence of trans-dominant inhibition of poly(ADP-ribosyl)ation on the rejoining kinetics of radiation-induced DNA double-strand breaks (DSB). MATERIALS AND METHODS: Stable transfectants of the SV40-transformed hamster cell line CO60 were used: COM3 cells contain a construct to overexpress the poly(ADP-ribose) polymerase (PARP-1) DNA-binding domain (DBD) when induced by dexamethasone, as well as a construct for the constitutive overexpression of the human glucocorticoid receptor (Hg0). COR3 are control cells containing only the Hg0 plasmid. DSB induction and rejoining in X-irradiated cells was assessed by DNA pulsed-field electrophoresis. RESULTS: DSB induction was identical in both cell lines and independent of the presence of dexamethasone. DSB rejoining kinetics was independent of dexamethasone in COR3 cells and identical to COM3 cells without dexamethasone. However, in COM3 cells treated with dexamethasone to induce PARP-1 DBD overexpression, the fast component of the rejoining kinetic was largely reduced, and residual fragmentation increased concomitant with the increased damage fraction in slow rejoining. CONCLUSIONS: The results indicate that inhibition of cellular PARP-1 does not affect the rate-limiting step of either fast or slow DSB rejoining. Rather, it appears that absence of poly(ADP-ribosyl)ation due to dominant negative PARP-1 expression induces a shift from rapid to slow DSB rejoining and by this mechanism PARP inhibition may increase the risk of repair failures.

Animals↗

PubMed1990

The yield of DNA double-strand breaks produced intracellularly by ionizing radiation: a review.

The mechanisms of radiation damage production are used to examine the following premises: (1) the number of DNA double-strand breaks per unit dose increases with dose; (2) cell type to cell type variations in yield of DNA dsb per dose occur. Two stages of damage production are identified as possible sources of damage yield modulation; numbers of OH. free radicals reacting with the target, and amount of chemical repair occurring on the target radicals. These factors are discussed in the light of the structures within which cellular DNA is packaged and the known rate constants for the reactions involved. It is concluded from our current knowledge that, in the presence of oxygen: (a) the number of DNA dsb is linearly related to dose, and (b) the yields of DNA damage per dose among cell types are constant. There is a caveat to the latter conclusion: the chromatin structure may be different in radiosensitive cell lines. In the absence of such a difference, variations in radiosensitivity with dose or with cell type are assigned to differences in repair speed and/or accuracy.

Cell Line↗