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PubMedDate not supplied

Mammalian X-ray-sensitive mutants which are defective in non-homologous (illegitimate) DNA double-strand break repair.

In all organisms multiple pathways to repair DNA double-strand breaks (DSB) have been identified. In mammalian cells DSB are repaired by two distinct pathways, homologous and non-homologous (illegitimate) recombination. X-ray-sensitive mutants have provided a tool for the identification and understanding of the illegitimate recombination pathway in mammalian cells. Two (sub-)pathways can be distinguished, the first mediated by DNA-PK-dependent protein kinase (DNA-PK), and the second directed by the hMre11/hRad50 complex. A variety of mutants impaired in DSB repair by illegitimate recombination, with mutations in Ku, DNA-PKcs, XRCC4 or nibrin, have been described. Herein, the characterization of these mutants with respect to the impaired cellular function and the molecular defect is provided. Further studies on these mutants, as well as on new mutants impaired in as-of-yet unidentified pathways, should be helpful to a better understanding of DSB repair and of the processes leading to genome instability and cancer.

Animals↗

PubMed2000

DNA double strand break repair in mammalian cells.

Human cells can process DNA double-strand breaks (DSBs) by either homology directed or non-homologous repair pathways. Defects in components of DSB repair pathways are associated with a predisposition to cancer. The products of the BRCA1 and BRCA2 genes, which normally confer protection against breast cancer, are involved in homology-directed DSB repair. Defects in another homology-directed pathway, single-strand annealing, are associated with genome instability and cancer predisposition in the Nijmegen breakage syndrome and a radiation-sensitive ataxia-telangiectasia-like syndrome. Many DSB repair proteins also participate in the signaling pathways which underlie the cell's response to DSBs.

Animals↗

PubMed2003

The cellular response to DNA double-strand breaks: defining the sensors and mediators.

The induction of DNA double-strand breaks (DSBs) culminates in the activation of cell cycle checkpoint responses and DNA repair machinery. The mechanism of DSB detection remains unclear although many candidate sensor proteins have been identified through cytologic, biochemical and genetic studies. In light of recent advances in our understanding of the cellular response to DSBs, we have proposed criteria for defining sensor proteins. We discuss the possible role of the Mre11 complex as a primary damage sensor and the complex relationship between DNA damage sensors, transducers and mediators.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Sex and the single (double-strand) break.

It has been known for some time that DNA double-strand breaks (DSBs) initiate homologous recombination during meiosis. Two recent studies show that the fate of a single DSB in yeast is strongly influenced by the presence of other breaks in the genome, hinting that cell-wide or chromosome-regional mechanisms control the outcome of DSB repair.

Chromosome Breakage↗

PubMed2000

Frequent chromosomal translocations induced by DNA double-strand breaks.

The faithful repair of DNA damage such as chromosomal double-strand breaks (DSBs) is crucial for genomic integrity. Aberrant repair of these lesions can result in chromosomal rearrangements, including translocations, which are associated with numerous tumours. Models predict that some translocations arise from DSB-induced recombination in differentiating lymphoid cell types or from aberrant repair of DNA damage induced by irradiation or other agents; however, a genetic system to study the aetiology of these events has been lacking. Here we use a mouse embryonic stem cell system to examine the role of DNA damage on the formation of translocations. We find that two DSBs, each on different chromosomes, are sufficient to promote frequent reciprocal translocations. The results are in striking contrast with interchromosomal repair of a single DSB in an analogous system in which translocations are not recovered. Thus, while interchromosomal DNA repair does not result in genome instability per se, the presence of two DSBs in a single cell can alter the spectrum of repair products that are recovered.

Animals↗

PubMed1992

Constraints on energy deposition and target size of multiply damaged sites associated with DNA double-strand breaks.

We suggest that the observed experimental data on relative double-strand break (dsb) yield as a function of radiation quality can act as valuable constraints in defining the type of energy deposition which causes this basic lesion in radiation biology. Both heavy-ion and alpha-particle data show sufficient trends for quantitative comparisons with calculation to be made. We use the technique of track-structure simulation and search for energy-deposition clusters (containing at least a given number of ionizations in a given diameter) whose relative frequencies (compared to sparsely ionizing radiation) correlate with the relative biological effects (RBEs) for dsb induction. We conclude that locally multiply damaged sites (LMDS) which cause dsb are probably energy depositions of at least two to five ionizations localized, respectively, in sites of diameters of 1-4 nm. Although our derived cluster sizes should be viewed in light of the quality of the experimental data and uncertainties in the computer simulations at the nanometre level, it is unlikely that these estimates of cluster sizes would change greatly.

Alpha Particles↗

PubMed1997

DNA double-strand breaks caused by replication arrest.

We report here that DNA double-strand breaks (DSBs) form in Escherichia coli upon arrest of replication forks due to a defect in, or the inhibition of, replicative DNA helicases. The formation of DSBs was assessed by the appearance of linear DNA detected by pulse-field gel electrophoresis. Processing of DSBs by recombination repair or linear DNA degradation was abolished by mutations in recBCD genes. Two E. coli replicative helicases were tested, Rep, which is essential in recBC mutants, and DnaB. The proportion of linear DNA increased up to 50% upon shift of rep recBTS recCTS cells to restrictive temperature. No increase in linear DNA was observed in the absence of replicating chromosomes, indicating that the formation of DSBs in rep strains requires replication. Inhibition of the DnaB helicase either by a strong replication terminator or by a dnaBTS mutation led to the formation of linear DNA, showing that blocked replication forks are prone to DSB formation. In wild-type E. coli, linear DNA was detected in the absence of RecBC or of both RecA and RecD. This reveals the existence of a significant amount of spontaneous DSBs. We propose that some of them may also result from the impairment of replication fork progression.

Bacterial Proteins↗

PubMed2000

Mechanisms of DNA double-strand break repair and their potential to induce chromosomal aberrations.

DNA double-strand breaks (DSB) are considered to be critical primary lesions in the formation of chromosomal aberrations. DSB may be induced by exogenous agents, such as ionizing radiation, but also occur spontaneously during cellular processes at quite significant frequencies. To repair this potentially lethal damage, eukaryotic cells have evolved a variety of repair pathways related to homologous and illegitimate recombination, also called non-homologous DNA end joining, which may induce small scale mutations and chromosomal aberrations. In this paper we review the major cellular sources of spontaneous DSB and the different homologous and illegitimate recombination repair pathways, with particular focus on their potential to induce chromosomal aberrations.

Animals↗

PubMed1993

Homologous recombination in plant cells is enhanced by in vivo induction of double strand breaks into DNA by a site-specific endonuclease.

Induction of double strand breaks (DSBs) is coupled to meiotic and mitotic recombination in yeast. We show that also in a higher eukaryote induction of DSBs is directly correlated with a strong enhancement of recombination frequencies. We cotransfected Nicotiana plumbaginifolia protoplasts with a plasmid carrying a synthetic I-SceI gene, coding for a highly sequence specific endonuclease, together with recombination substrates carrying an I-SceI-site adjacent to their homologous sequences. We measured efficiencies of extrachromosomal recombination, using a well established transient beta-glucuronidase (GUS) assay. GUS enzyme activities were strongly increased when a plasmid carrying the I-SceI gene in sense but not in antisense orientation with respect to the promoter was included in the transfections. The in vivo induced DSBs were detected in the recombination substrates by Southern blotting, demonstrating that the yeast enzyme is functional in plant cells. At high ratios of transfected I-SceI-genes to I-SceI-sites the majority of the I-SceI-sites in the recombination substrates are cleaved, indicating that the induction of the DSBs is the rate limiting step in the described recombination reaction. These results imply that in vivo induction of transient breaks at specific sites in the plant genome could allow foreign DNA to be targeted to these sites via homologous recombination.

Base Sequence↗

PubMed1995

A modified single-strand annealing model best explains the joining of DNA double-strand breaks mammalian cells and cell extracts.

The joining of DNA double-strand breaks in vivo is frequently accompanied by the loss of a few nucleotides at the junction between the interacting partners. In vitro systems mimic this loss and, on detailed analysis, have suggested two models for the mechanism of end-joining. One invokes the use of extensive homologous side-by-side alignment of the partners prior to joining, while the other proposes the use of small regions of homology located at or near the terminus of the interacting molecules. to discriminate between these two models, assays were conducted both in vitro and in vivo with specially designed substrates. In vitro, molecules with limited terminal homology were capable of joining, but analysis of the junctions suggested that the mechanism employed the limited homology available. In vivo, the substrates with no extensive homology end-joined with equal efficiency to those with extensive homology in two different topological arrangements. Taken together, these results suggest that extensive homology is not a prerequisite for efficient end-joining, but that small homologies close to the terminus are used preferentially, as predicted by the modified single-strand annealing model.

Base Sequence↗

PubMed1994

Homotypic and heterotypic protein associations control Rad51 function in double-strand break repair.

Rad51 is essential for efficient repair of DNA double-strand breaks (DSBs) and recombination in Saccharomyces cerevisiae. Here, we examine Rad51 protein-protein interactions and their biological significance. GAL4 two-hybrid fusion analysis demonstrated that the amino-terminal region of Rad51 mediates both a strong Rad51:Rad51 self-association and a Rad51:Rad52 interaction. Several Rad51 variants were characterized that imparted DSB repair defects; these defects appear to result from Rad51 protein-protein interactions. First, a rad51 allele bearing a missense mutation in the consensus ATP-binding sequence disrupted DSB repair in wild-type yeast. The effect of this allele was dependent on the presence of wild-type Rad51 because MMS sensitivity of rad51 delta strains were not increased by its expression. Second, we identified a highly conserved RAD51 homolog from Kluyveromyces lactis (KlRAD51) that only partially complemented rad51 delta strains and impaired DSB repair in wild-type S. cerevisiae. Third, fusions of Gal4 domains to Rad51 disrupted DSB repair in a manner that required the presence of either Rad51 or Rad52. Because K. lactis RAD51 and RAD52 did not complement a S. cerevisiae rad51 delta rad52 delta strain, Rad51-Rad52 functions appear to be mediated through additional components. Thus, multiple types of Rad51 protein interactions, including self-association, appear to be important for DSB repair.

Alleles↗

PubMed1997

Chromosomal double-strand breaks induce gene conversion at high frequency in mammalian cells.

Double-strand breaks (DSBs) stimulate chromosomal and extrachromosomal recombination and gene targeting. Transcription also stimulates spontaneous recombination by an unknown mechanism. We used Saccharomyces cerevisiae I-SceI to stimulate recombination between neo direct repeats in Chinese hamster ovary (CHO) cell chromosomal DNA. One neo allele was controlled by the dexamethasone-inducible mouse mammary tumor virus promoter and inactivated by an insertion containing an I-SceI site at which DSBs were introduced in vivo. The other neo allele lacked a promoter but carried 12 phenotypically silent single-base mutations that create restriction sites (restriction fragment length polymorphisms). This system allowed us to generate detailed conversion tract spectra for recipient alleles transcribed at high or low levels. Transient in vivo expression of I-SceI increased homologous recombination 2,000- to 10,000-fold, yielding recombinants at frequencies as high as 1%. Strikingly, 97% of these products arose by gene conversion. Most products had short, bidirectional conversion tracts, and in all cases, donor neo alleles (i.e., those not suffering a DSB) remained unchanged, indicating that conversion was fully nonreciprocal. DSBs in exogenous DNA are usually repaired by end joining requiring little or no homology or by nonconservative homologous recombination (single-strand annealing). In contrast, we show that chromosomal DSBs are efficiently repaired via conservative homologous recombination, principally gene conversion without associated crossing over. For DSB-induced events, similar recombination frequencies and conversion tract spectra were found under conditions of low and high transcription. Thus, transcription does not further stimulate DSB-induced recombination, nor does it appear to affect the mechanism(s) by which DSBs induce gene conversion.

Animals↗

PubMed2005

RAD-51-dependent and -independent roles of a Caenorhabditis elegans BRCA2-related protein during DNA double-strand break repair.

The BRCA2 tumor suppressor is implicated in DNA double-strand break (DSB) repair by homologous recombination (HR), where it regulates the RAD51 recombinase. We describe a BRCA2-related protein of Caenorhabditis elegans (CeBRC-2) that interacts directly with RAD-51 via a single BRC motif and that binds preferentially to single-stranded DNA through an oligonucleotide-oligosaccharide binding fold. Cebrc-2 mutants fail to repair meiotic or radiation-induced DSBs by HR due to inefficient RAD-51 nuclear localization and a failure to target RAD-51 to sites of DSBs. Genetic and cytological comparisons of Cebrc-2 and rad-51 mutants revealed fundamental phenotypic differences that suggest a role for Cebrc-2 in promoting the use of an alternative repair pathway in the absence of rad-51 and independent of nonhomologous end joining (NHEJ). Unlike rad-51 mutants, Cebrc-2 mutants also accumulate RPA-1 at DSBs, and abnormal chromosome aggregates that arise during the meiotic prophase can be rescued by blocking the NHEJ pathway. CeBRC-2 also forms foci in response to DNA damage and can do so independently of rad-51. Thus, CeBRC-2 not only regulates RAD-51 during HR but can also function independently of rad-51 in DSB repair processes.

Amino Acid Motifs↗

PubMed2004

Increased repair of gamma-induced DNA double-strand breaks at lower dose-rate in CHO cells.

DNA double-strand breaks (DSBs) are highly cell damaging. We asked whether for a given dose a longer irradiation time would be advantageous for the repair of DSBs. Varying the gamma-irradiation dose and its delivery time (0.05 Gy/min low dose-rate (LDR) compared with 3.5 Gy/min high dose-rate), confluent Chinese hamster ovary cells (CHO-K1) and Ku80 mutant cells (xrs-6) deficient in nonhomologous end-joining (NHEJ) were irradiated in agarose plugs at room temperature using a cesium-137 gamma-ray source. We used pulsed-field gel electrophoresis (PFGE) to measure DSBs in terms of the fraction of activity released (FAR). At LDR, one third of DSBs were repaired in CHO-K1 but not in xrs-6 cells, indicating the involvement of NHEJ in the repair of gamma-induced DSBs at a prolonged irradiation incubation time. To improve DSB measurements, we introduced in our PFGE protocol an antioxidant at the cell lysis step, thus avoiding free-radical side reactions on DNA and spurious DSBs. Addition of the metal chelator deferoxamine (DFO) decreased more efficiently the basal DSB level than did reduced glutathione (GSH), showing that measuring DSBs in the absence of DFO reduces precision and underestimates the role of NHEJ in the dose-rate effect on DSB yield.

Animals↗

PubMed2005

MLH1 and MSH2 promote the symmetry of double-strand break repair events at the HIS4 hotspot in Saccharomyces cerevisiae.

Double-strand breaks (DSBs) initiate meiotic recombination. The DSB repair model predicts that both genetic markers spanning the DSB should be included in heteroduplex DNA and be detectable as non-Mendelian segregations (NMS). In experiments testing this, a significant fraction of events do not conform to this prediction, as only one of the markers displays NMS (one-sided events). Two explanations have been proposed to account for the discrepancies between the predictions and experimental observations. One suggests that two-sided events are the norm but are "hidden" as heteroduplex repair frequently restores the parental configuration of one of the markers. Another explanation posits that one-sided events reflect events in which heteroduplex is formed predominantly on only one side of the DSB. In the absence of heteroduplex repair, the first model predicts that two-sided events would be revealed at the expense of one-sided events, while the second predicts no effect on the distribution of events when heteroduplex repair is lost. We tested these predictions by deleting the DNA mismatch repair genes MSH2 or MLH1 and analyzing the proportion of two-sided events. Unexpectedly, the results do not match the predictions of either model. In both mlh1Delta and msh2Delta, the proportion of two-sided events is significantly decreased relative to wild type. These observations can be explained in one of two ways. Either Msh2p/Mlh1p-independent mispair removal leads to restoration of one of the markers flanking the DSB site or Msh2p/Mlh1p actively promote two-sided events.

Adaptor Proteins, Signal Transducing↗

PubMed2006

ATR-Chk1 axis protects BCR/ABL leukemia cells from the lethal effect of DNA double-strand breaks.

BCR/ABL-positive leukemia cells accumulated more replication-dependent DNA double-strand breaks (DSBs) than normal counterparts after treatment with cisplatin and mitomycin C (MMC, as assessed by pulse field gel electrophoresis (PFGE) and neutral comet assay. In addition, leukemia cells could repair these lesions more efficiently than normal cells and eventually survive genotoxic treatment. Elevated levels of drug-induced DSBs in leukemia cells were associated with higher activity of ATR kinase, and enhanced phosphorylation of histone H2AX on serine 139 (gamma-H2AX). gamma-H2AX eventually started to disappear in BCR/ABL cells, while continued to increase in parental cells. In addition, the expression and ATR-mediated phosphorylation of Chk1 kinase on serine 345 were often more abundant in BCR/ABL-positive leukemia cells than normal counterparts after genotoxic treatment. Inhibition of ATR kinase by caffeine but not Chk1 kinase by indolocarbazole inhibitor, SB218078 sensitized BCR/ABL leukemia cells to MMC in a short-term survival assay. Nevertheless, both caffeine and SB218078 enhanced the genotoxic effect of MMC in a long-term clonogenic assay. This effect was associated with the abrogation of transient accumulation of leukemia cells in S and G2/M cell cycle phases after drug treatment. In conclusion, ATR-Chk1 axis was strongly activated in BCR/ABL-positive cells and contributed to the resistance to DNA cross-linking agents causing numerous replication-dependent DSBs.

Antineoplastic Agents↗

PubMed1984

Interpretation of the shape of survival curves in terms of induction and repair/misrepair of DNA double-strand breaks.

Evidence is presented that in yeast cells one DNA double-strand break (dsb) may be considered as one potentially lethal lesion (PLL). Using a temperature conditional radiosensitive diploid yeast mutant (rad 54-3) it is demonstrated that the shoulder of survival curves, for cells plated immediately, is due to repair of dsb (PLL) within a restricted time period. Split dose experiments with the mutant rad 54-3 show that the reappearance of a shoulder is observed when two conditions are met: (1) repair of dsb (PLL) during the time interval between doses and (2) repair of mainly those dsb (PLL) which are induced by the second dose on the nutrient agar plate. Irradiation of wild type yeast cells at high dose rate followed by liquid holding treatment for 72 h (delayed plating, DP) or at low dose rate show that the bending of DP-survival curves is due to the accumulation of dsb (PLL) leading to lethal lesions probably by misrepair of dsb.

DNA↗

PubMed1998

Identification of genes involved in repair of DNA double-strand breaks in mammalian cells.

At least two mechanisms of DNA double-strand break (DSB) repair operate in mammalian cells. Homologous recombination, which plays a major role in lower organisms, plays a less significant role in higher organisms. In contrast, the majority of DSBs in mammalian cells are rejoined by a mechanism, termed non-homologous end joining (NHEJ), that does not depend upon extensive regions of homology. This process is also used to rejoin site-specific DSBs introduced during V(D)J recombination. From the analysis of defective rodent mutants, four proteins (Ku70, Ku80, DNA-PKcs and Xrcc4) that function in this process in mammalian cells have been identified. DNA ligase IV is also strongly implicated since it associates strongly with XRCC4, and since DNA ligase IV-deficient yeast are defective in their ability to carry out NHEJ. In S. cerevisiae, Sir2p, Sir3p and Sir4p, three proteins required for transcriptional silencing, are also required for NHEJ. Additionally, the yeast mutants, xrs2, rad50 and mre11, which are defective in meiotic recombination, are also defective in NHEJ. Here I review the evidence implicating these proteins as functioning in NHEJ and discuss their properties and role in other pathways. The significance of DSB repair to clinical radiosensitivity and human disorders is also evaluated.

Animals↗