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PubMed1992

Molecular mechanisms of the formation of DNA double-strand breaks and induction of genomic rearrangements.

The probability that damage occurs in closely opposed sites on complementary DNA strands increases when DNA is heavily modified with mutagenic agents. Enzymatic excision of the opposite lesions produces DNA double-strand breaks which give rise to genomic rearrangements (deletions, insertions, etc.). Plasmid systems were developed for studying chemical lesions leading to double-strand breaks and the fate of broken plasmid molecules within bacterial cells. Deletions result from the base-pairing of fortuitously located direct repeats flanking the DNA broken ends; as a consequence, the latter are joined, while the DNA fragment between the direct repeats is deleted. Genomic rearrangements arise during the repair of the DNA double-strand breaks, and both events are due to similar repair enzymes which maintain the integrity of the DNA primary structure when conditions are not stressful. A number of genomic rearrangements and point mutations seem to be predetermined by the DNA primary structure.

Base Sequence↗

PubMed1975

The repair of DNA double-strand breaks in mammalian cells and the organization of the DNA in their chromosomes.

The molecular weight of native DNA has been accurately determined by the use of a semiautomated sucrose gradient system. A mondisperse size distribution (speed dependence free) of eighth-of-a-chromatid pieces [1.7 S 10(10) daltons, with 95% confidence (fiducial) limits of +/- 48%] was found. This size has been confirmed by viscoelastometry. Ionizing radiation rapidly breaks each of these pieces into about 21 subunits (again monodisperse) of 8 X 10(8) daltons each. With increaseing dose (greater than 2 krad) the subunits are themselves randomly broken down into even smaller pieces. Postirradiation incubation at 37 degrees C permits the cells to repair both DNA double-strand breaks and intersubunit linkages at the same dose-independent rate (T37) of about 55 min, the same rate as found in Micrococcus radiodurans. The repair data are compatible with a first-order-kinetics repair system, analogous to the post-UV excision-repair system, which becomes saturated at high doses (greater than 60 krad). Specially constructed "enzyme" gradients show that the linkages contain at least two protein molecules each covalently bound to the end of a subunit and linking the subunits together by a disulfide bond(s). Correlation of cell survival and DNA break kinetics yields two possible models. These are that the two-thirds of the lethal events which are due to improperly or unrepaired double-strand breaks result from either (1) a misrepair frequency of 3.5 X 10(-3) (rather high for a mutation frequency) or (2) the induction of a double-strand break in a single eighth-of-a-chromatid unit which is essential for survival but which cannot be repaired, possibly because the unit contains the double-strand break repair system gene(s).

Cell Line↗

PubMed1996

End-joining of free radical-mediated DNA double-strand breaks in vitro is blocked by the kinase inhibitor wortmannin at a step preceding removal of damaged 3' termini.

Both mammalian cells and Xenopus eggs possess activities for the joining of nonhomologous DNA ends, and such activities may play a major role in double-strand break repair. In order to dissect the biochemical processing of breaks with oxidatively modified ends, vectors containing various site-specific double-strand breaks with 3'-phosphoglycolate termini were constructed and treated with Xenopus egg extracts. These vectors were rejoined by the extracts at rates 30-100 times slower than comparable 3'-hydroxyl vectors. Vectors with blunt or cohesive 3'-phosphoglycolate ends yielded single repair products corresponding to simple phosphoglycolate removal followed by ligation, while a vector with mismatched ends was also rejoined but yielded a mixture of products. Addition of the kinase inhibitors wortmannin and dimethylaminopurine not only blocked rejoining, but also suppressed phosphoglycolate removal, implying an early, essential, kinase-dependent restriction point in the pathway. The results suggest that double-strand breaks with oxidatively modified ends are repaired in Xenopus eggs by a highly conservative and stringently regulated end-joining pathway, in which all biochemical processing of the breaks is contingent on both end alignment and a specific phosphorylation event. Several lines of indirect evidence suggest DNA-dependent protein kinase as a likely candidate for effecting this phosphorylation.

Androstadienes↗

PubMed2001

When X-ray-inducible proteins meet DNA double strand break repair.

Cellular responses to ionizing radiation (IR) include (a) activation of signal transduction enzymes; (b) stimulation of DNA repair, most notably DNA double strand break (DSB) repair by homologous or nonhomologous recombinatorial pathways; (c) activation of transcription factors and subsequent IR-inducible transcript and protein changes; (d) cell cycle checkpoint delays in G(1), S, and G(2) required for repair or for programmed cell death of severely damaged cells; (e) activation of zymogens needed for programmed cell death (although IR is a poor inducer of such responses in epithelial cells); and (f) stimulation of IR-inducible proteins that may mediate bystander effects influencing signal transduction, DNA repair, angiogenesis, the immune response, late responses to IR, and possibly adaptive survival responses. The overall response to IR depends on the cell's inherent genetic background, as well as its ability to biochemically and genetically respond to IR-induced damage. To improve the anti-tumor efficacy of IR, our knowledge of these pleiotropic responses must improve. The most important process for the survival of a tumor cell following IR is the repair of DNA double strand breaks (DSBs). Using yeast two-hybrid analyses along with other molecular and cellular biology techniques, we cloned transcripts/proteins that are involved in, or presumably affect, nonhomologous DNA double strand break end-joining (NHEJ) repair mediated by the DNA-PK complex. Using Ku70 as bait, we isolated a number of Ku-binding proteins (KUBs). We identified the first X-ray-inducible transcript/protein (xip8, Clusterin (CLU)) that associates with DNA-PK. A nuclear form of CLU (nCLU) prevented DNA-PK-mediated end joining, and stimulated cell death in response to IR or when overexpressed in the absence of IR. Structure-function analyses using molecular and cellular (including green fluorescence-tagged protein trafficking) biology techniques showed that nCLU appears to be an inactive protein residing in the cytoplasm of epithelial cells. Following IR injury, nCLU levels increase and an as yet undefined posttranslational modification appears to alter the protein, exposing nuclear localization sequences (NLSs) and coiled-coil domains. The modified protein translocates to the nucleus and triggers cell death, presumably through its interaction specifically with Ku70. Understanding nCLU responses, as well as the functions of the KUBs, will be important for understanding DSB repair. Knowledge of DSB repair may be used to improve the antitumor efficacy of IR, as well as other chemotherapeutic agents.

Cell Survival↗

PubMed1995

Complex formation in yeast double-strand break repair: participation of Rad51, Rad52, Rad55, and Rad57 proteins.

The repair of DNA double-strand breaks in Saccharomyces cerevisiae requires genes of the RAD52 epistasis group, of which RAD55 and RAD57 are members. Here, we show that the x-ray sensitivity of rad55 and rad57 mutant strains is suppressible by overexpression of RAD51 or RAD52. Virtually complete suppression is provided by the simultaneous overexpression of RAD51 and RAD52. This suppression occurs at 23 degrees C, where these mutants are more sensitive to x-rays, as well as at 30 degrees C and 36 degrees C. In addition, a recombination defect of rad55 and rad57 mutants is similarly suppressed. Direct in vivo interactions between the Rad51 and Rad55 proteins, and between Rad55 and Rad57, have also been identified by using the two-hybrid system. These results indicate that these four proteins constitute part of a complex, a "recombinosome," to effect the recombinational repair of double-strand breaks.

Adenosine Triphosphatases↗

PubMed1993

Induction of oxidative single- and double-strand breaks in DNA by ferric citrate.

The relative risk of primary hepatocellular carcinoma in genetic hemochromatosis (GH) is estimated at over 200 times as that of control populations. Recently, ferric ion chelated to citrate (Fe-citrate) was identified as the major non-transferrin-bound iron in the serum of GH patients. We investigated whether low concentration of Fe-citrate plus reductant could damage supercoiled plasmid DNA under physiological pH and ionic strength. Incubation of Fe-citrate with either H2O2, L-ascorbate, or L-cysteine induced single- and double-strand breaks in supercoiled plasmid pZ189 in a concentration- and time-dependent fashion. DNA strand breaks produced by Fe-citrate plus H2O2 increased at reduced pH (< or = 6.9). Catalase and free radical scavengers inhibited the DNA breakage produced by Fe-citrate in combination with each reductant, suggesting that H2O2 and finally .OH are responsible DNA damaging species. The catalytic ability of Fe-citrate to induce DNA strand breaks, particularly double-strand breaks (DSBs), may contribute to the carcinogenic processes observed in GH.

Ascorbic Acid↗

PubMed1990

Multiple pathways of DNA double-strand break processing in a mutant Indian muntjac cell line.

DNA break processing is compared in the Indian muntjac cell lines, SVM and DM. The initial frequencies and resealing of X-ray generated single- and double-strand breaks are similar in the two cell lines. Inhibiting the repair of UV damage leads to greater double-strand breakage in SVM than in DM, and some of these breaks are not repaired; however, repair-associated single-strand breakage and resealing are normal. Dimethylsulfate also induces excess double-strand breakage in SVM, and these breaks are irreparable. Restricted plasmids are reconstituted correctly in SVM at approximately 30% of the frequency observed in DM. Thus SVM has a reduced capacity to repair certain types of double-strand break. This defect is not due to a DNA ligase deficiency. We conclude that DNA double-strand breaks are repaired by a variety of pathways within mammalian cells and that the structure of the break or its mode of formation determines its subsequent fate.

Animals↗

PubMed2000

Ionizing radiation enhances double-strand-break repair in rapamycin-treated ataxia telangiectasia lymphoblasts.

PURPOSE: Unlike normal cells, ataxia telangiectasia (A-T) cells do not exhibit enhanced double-strand-break (dsb)-repair fidelity after ionizing radiation (IR) exposure. DNA-repair induction and other responses to IR are mediated by signalling pathways that are defective in A-T cells. Ataxia telangiectasia mutated (ATM) protein, the mutated protein in A-T cells, is homologous to members of the P13-kinase, including the rapamycin target FRAP. Rapamycin kills normal cells more readily in normal than in A-T cells, and inhibits the FRAP target p70 S6 kinase (p70S6K) more readily in normal than in A-T cells. Also, PHAS-1, another FRAP target, may also be a substrate for ATM. Because ATM plays a role in the response of mammalian cells to rapamycin, the effect of rapamycin on IR enhancement of dsb repair was investigated. MATERIALS AND METHODS: Double-strand-break repair by normal and A-T lymphoblasts, either untreated or treated with rapamycin and y-irradiated, was analysed using shuttle pZ189 containing a dsb. RESULTS: Double-strand-break-rejoining fidelity in linear plasmids (linDNA) processed in either untreated or rapamycin-treated normal cells increased about twofold if transfection occurred immediately after host irradiation. In contrast, radiation did not increase or decrease the repair fidelity by untreated A-T host cells. But, like normal hosts, dsb-repair fidelity improved twofold in A-T hosts treated with rapamycin. Treatment with the P13-kinase inhibitor LY294002 did not alter dsb-rejoining fidelity in normal or in A-T hosts. CONCLUSIONS: These findings demonstrate that rapamycin does not affect IR enhancement of dsb-repair fidelity in normal cells but restores this phenomenon in A-T lymphoblasts, and suggests the involvement of a rapamycin-sensitive pathway in radiation-enhanced dsb repair.

Cell Line↗

PubMed2005

Recent advances in understanding of the DNA double-strand break repair machinery of plants.

Living cells suffer numerous and varied alterations of their genetic material. Of these, the DNA double-strand break (DSB) is both particularly threatening and common. Double-strand breaks arise from exposure to DNA damaging agents, but also from cell metabolism-in a fortuitous manner during DNA replication or repair of other kinds of lesions and in a programmed manner, for example during meiosis or V(D)J gene rearrangement. Cells possess several overlapping repair pathways to deal with these breaks, generally designated as genetic recombination. Genetic and biochemical studies have provided considerable amounts of data about the proteins involved in recombination processes and their functions within these processes. Although they have long played a key role in building understanding of genetics, relatively little is known at the molecular level of the genetic recombination processes in plants. The use of reverse genetic approaches and the public availability of sequence tagged mutants in Arabidopsis thaliana have led to increasingly rapid progress in this field over recent years. The rapid progress of studies of recombination in plants is obviously not limited to the DSB repair machinery as such and we ask readers to understand that in order to maintain the focus and to rest within a reasonable length, we present only limited discussion of the exciting advances in the of plant meiosis field, which require a full review in their own right . We thus present here an update on recent advances in understanding of the DSB repair machinery of plants, focussing on Arabidopsis and making a particular effort to place these in the context of more general of understanding of these processes.

Adenosine Triphosphatases↗

PubMed1996

Disruption of DNA-PK in Ku80 mutant xrs-6 and the implications in DNA double-strand break repair.

The Chinese hamster ovary (CHO) mutant cell line xrs-6C is highly sensitive to radiation and is deficient in DNA double-strand break (DSB) repair. The repair defect of xrs-6C is complemented by the human DSB repair gene designated as XRCC5. This gene was recently identified as Ku80, which encodes the human autoantigen protein Ku p80. Ku80 protein forms heterodimer with the Ku70 subunit to form a complex that possesses a DNA end-binding activity. Ku70/Ku80 heterodimer can recruit the catalytic p350 subunit of the DNA-dependent protein kinase. It is demonstrated here that, while the Ku70 mRNA expression is normal in the xrs-6C mutant, Ku70 protein is undetectable. However, introduction of human Ku80 gene into the mutant lead to increased expression of Ku70 protein and restored Ku70 binding to DNA ends, suggesting that mutation of the Ku80 gene affected the formation of Ku70/Ku80 dimers and the stability of the Ku70 protein. We also demonstrated that, although p350 protein expression in the mutants was unaffected, the capacity of p350 to bind to DNA ends was impaired in the mutants. After introduction of the human Ku80 into the mutant, the association of p350 with DNA end was restored, accompanied by recovery in cell survival and DNA double-strand break repair. The results in this report show that mutation of the Ku80 gene disrupts formation of the Ku70/Ku80 dimer and compromises the ability of Ku protein to recruit the DNA-PK p350 subunit to DNA double-strand breaks, causing a dysfunction of DNA DSB repair in the cell.

Animals↗

PubMed2006

DNA double-strand break repair: a relentless hunt uncovers new prey.

A major pathway for repair of DNA double-strand breaks is nonhomologous end-joining (NHEJ). In this issue of Cell, and report the discovery of a new NHEJ factor called Cernunnos-XLF. Both groups report that this protein is mutated in a rare inherited human syndrome characterized by severe immunodeficiency, developmental delay, and hypersensitivity to agents that cause DNA double-strand breaks.

Animals↗

PubMed2005

Analysis of global gene expression and double-strand-break formation in DNA adenine methyltransferase- and mismatch repair-deficient Escherichia coli.

DNA adenine methylation by DNA adenine methyltransferase (Dam) in Escherichia coli plays an important role in processes such as DNA replication initiation, gene expression regulation, and mismatch repair. In addition, E. coli strains deficient in Dam are hypersensitive to DNA-damaging agents. We used genome microarrays to compare the transcriptional profiles of E. coli strains deficient in Dam and mismatch repair (dam, dam mutS, and mutS mutants). Our results show that >200 genes are expressed at a higher level in the dam strain, while an additional mutation in mutS suppresses the induction of many of the same genes. We also show by microarray and semiquantitative real-time reverse transcription-PCR that both dam and dam mutS strains show derepression of LexA-regulated SOS genes as well as the up-regulation of other non-SOS genes involved in DNA repair. To correlate the level of SOS induction and the up-regulation of genes involved in recombinational repair with the level of DNA damage, we used neutral single-cell electrophoresis to determine the number of double-strand breaks per cell in each of the strains. We find that dam mutant E. coli strains have a significantly higher level of double-strand breaks than the other strains. We also observe a broad range in the number of double-strand breaks in dam mutant cells, with a minority of cells showing as many as 10 or more double-strand breaks. We propose that the up-regulation of recombinational repair in dam mutants allows for the efficient repair of double-strand breaks whose formation is dependent on functional mismatch repair.

Adenosine Triphosphatases↗

PubMed1992

In vitro repair of double-strand breaks accompanied by recombination in bacteriophage T7 DNA.

A double-strand break in a bacteriophage T7 genome significantly reduced the ability of that DNA to produce viable phage when the DNA was incubated in an in vitro DNA replication and packaging system. When a homologous piece of T7 DNA (either a restriction fragment or T7 DNA cloned into a plasmid) that was by itself unable to form a complete phage was included in the reaction, the break was repaired to the extent that many more viable phage were produced. Moreover, repair could be completed even when a gap of about 900 nucleotides was put in the genome by two nearby restriction cuts. The repair was accompanied by acquisition of a genetic marker that was present only on the restriction fragment or on the T7 DNA cloned into a plasmid. These data are interpreted in light of the double-strand gap repair mode of recombination.

Cell-Free System↗

PubMed1999

Lack of dependence on p53 for DNA double strand break repair of episomal vectors in human lymphoblasts.

The p53 tumor suppressor gene has been shown to be involved in a variety of repair processes, and recent findings have suggested that p53 may be involved in DNA double strand break repair in irradiated cells. The role of p53 in DNA double strand break repair, however, has not been fully investigated. In this study, we have constructed a novel Epstein-Barr virus (EBV)-based shuttle vector, designated as pZEBNA, to explore the influence of p53 on DNA strand break repair in human lymphoblasts, since EBV-based vectors do not inactivate the p53 pathway. We have compared plasmid survival of irradiated, restriction enzyme linearized, and calf intestinal alkaline phosphatase (CIP)-treated pZEBNA with a Simian virus 40 (SV40)-based shuttle vector, pZ189, in TK6 (wild-type p53) and WTK1 (mutant p53) lymphoblasts and determined that p53 does not modulate DNA double strand break repair in these cell lines.

Cell Line↗

PubMed2002

BRCA1 facilitates microhomology-mediated end joining of DNA double strand breaks.

BRCA1 is critical for the maintenance of genomic stability, in part through its interaction with the Rad50.Mre11.Nbs1 complex, which occupies a central role in DNA double strand break repair mediated by nonhomologous end joining (NHEJ) and homologous recombination. BRCA1 has been shown to be required for homology-directed recombination repair. However, the role of BRCA1 in NHEJ, a critical pathway for the repair of double strand breaks and genome stability in mammalian cells, remains elusive. Here, we established a pair of mouse embryonic fibroblasts (MEFs) derived from 9.5-day-old embryos with genotypes Brca1(+/+):p53(-/-) or Brca1(-/-):p53(-/-). The Brca1(-/-):p53(-/-) MEFs appear to be extremely sensitive to ionizing radiation. The contribution of BRCA1 in NHEJ was evaluated in these cells using three different assay systems. First, transfection of a linearized plasmid in which expression of the reporter gene required precise end joining indicated that Brca1(-/-) MEFs display a moderate deficiency when compared with Brca1(+/+) cells. Second, using a retrovirus infection assay dependent on NHEJ, a 5-10-fold reduction in retroviral integration efficiency was observed in Brca1(-/-) MEFs when compared with the Brca1(+/+) MEFs. Third, Brca1(-/-) MEFs exhibited a 50-100-fold deficiency in microhomology-mediated end-joining activity of a defined chromosomal DNA double strand break introduced by a rare cutting endonuclease I-SceI. These results provide evidence that Brca1 has an essential role in microhomology-mediated end joining and suggest a novel molecular basis for its caretaker role in the maintenance of genome integrity.

Adenoviridae↗

PubMed2006

The structure-specific endonuclease Mus81-Eme1 promotes conversion of interstrand DNA crosslinks into double-strands breaks.

Repair of interstrand crosslinks (ICLs) requires multiple-strand incisions to separate the two covalently attached strands of DNA. It is unclear how these incisions are generated. DNA double-strand breaks (DSBs) have been identified as intermediates in ICL repair, but enzymes responsible for producing these intermediates are unknown. Here we show that Mus81, a component of the Mus81-Eme1 structure-specific endonuclease, is involved in generating the ICL-induced DSBs in mouse embryonic stem (ES) cells in S phase. Given the DNA junction cleavage specificity of Mus81-Eme1 in vitro, DNA damage-stalled replication forks are suitable in vivo substrates. Interestingly, generation of DSBs from replication forks stalled due to DNA damage that affects only one of the two DNA strands did not require Mus81. Furthermore, in addition to a physical interaction between Mus81 and the homologous recombination protein Rad54, we show that Mus81(-/-) Rad54(-/-) ES cells were as hypersensitive to ICL agents as Mus81(-/-) cells. We propose that Mus81-Eme1- and Rad54-mediated homologous recombination are involved in the same DNA replication-dependent ICL repair pathway.

Animals↗

PubMed2004

Saccharomyces cerevisiae Sin3p facilitates DNA double-strand break repair.

There are two main pathways in eukaryotic cells for the repair of DNA double-strand breaks: homologous recombination and nonhomologous end joining. Because eukaryotic genomes are packaged in chromatin, these pathways are likely to require the modulation of chromatin structure. One way to achieve this is by the acetylation of lysine residues on the N-terminal tails of histones. Here we demonstrate that Sin3p and Rpd3p, components of one of the predominant histone deacetylase complexes of Saccharomyces cerevisiae, are required for efficient nonhomologous end joining. We also show that lysine 16 of histone H4 becomes deacetylated in the proximity of a chromosomal DNA double-strand break in a Sin3p-dependent manner. Taken together, these results define a role for the Sin3p/Rpd3p complex in the modulation of DNA repair.

Base Sequence↗

PubMed2002

Spatial distribution and yield of DNA double-strand breaks induced by 3-7 MeV helium ions in human fibroblasts.

Accelerated helium ions with mean energies at the target location of 3-7 MeV were used to simulate alpha-particle radiation from radon daughters. The experimental setup and calibration procedure allowed determination of the helium-ion energy distribution and dose in the nuclei of irradiated cells. Using this system, the induction of DNA double-strand breaks and their spatial distributions along DNA were studied in irradiated human fibroblasts. It was found that the apparent number of double-strand breaks as measured by a standard pulsed-field gel assay (FAR assay) decreased with increasing LET in the range 67-120 keV/microm (corresponding to the energy of 7-3 MeV). On the other hand, the generation of small and intermediate-size DNA fragments (0.1-100 kbp) increased with LET, indicating an increased intratrack long-range clustering of breaks. The fragment size distribution was measured in several size classes down to the smallest class of 0.1-2 kbp. When the clustering was taken into account, the actual number of DNA double-strand breaks (separated by at least 0.1 kbp) could be calculated and was found to be in the range 0.010-0.012 breaks/Mbp Gy(-1). This is two- to threefold higher than the apparent yield obtained by the FAR assay. The measured yield of double-strand breaks as a function of LET is compared with theoretical Monte Carlo calculations that simulate the track structure of energy depositions from helium ions as they interact with the 30-nm chromatin fiber. When the calculation is performed to include fragments larger than 0.1 kbp (to correspond to the experimental measurements), there is good agreement between experiment and theory.

Cells, Cultured↗