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PubMed1999

DNA double-strand break repair proteins are required to cap the ends of mammalian chromosomes.

Recent findings intriguingly place DNA double-strand break repair proteins at chromosome ends in yeast, where they help maintain normal telomere length and structure. In the present study, an essential telomere function, the ability to cap and thereby protect chromosomes from end-to-end fusions, was assessed in repair-deficient mouse cell lines. By using fluorescence in situ hybridization with a probe to telomeric DNA, spontaneously occurring chromosome aberrations were examined for telomere signal at the points of fusion, a clear indication of impaired end-capping. Telomeric fusions were not observed in any of the repair-proficient controls and occurred only rarely in a p53 null mutant. In striking contrast, chromosomal end fusions that retained telomeric sequence were observed in nontransformed DNA-PK(cs)-deficient cells, where they were a major source of chromosomal instability. Metacentric chromosomes created by telomeric fusion became even more abundant in these cells after spontaneous immortalization. Restoration of repair proficiency through transfection with a functional cDNA copy of the human DNA-PK(cs) gene reduced the number of fusions compared with a negative transfection control. Virally transformed cells derived from Ku70 and Ku80 knockout mice also displayed end-to-end fusions. These studies demonstrate that DNA double-strand break repair genes play a dual role in maintaining chromosomal stability in mammalian cells, the known role in repairing incidental DNA damage, as well as a new protective role in telomeric end-capping.

Animals↗

PubMed2006

Links between DNA double strand break repair and breast cancer: accumulating evidence from both familial and nonfamilial cases.

DNA double strand break (DSB) repair dysfunction increases the risk of familial and sporadic breast cancer. Advances in the understanding of genetic predisposition to breast cancer have also been made by screening naturally occurring polymorphisms. These studies revealed that subtle defects in DNA repair capacity arising from low-penetrance genes, or combinations thereof, are modified by other genetically determined or environmental risk factors and correlate to breast cancer risk. Overexpression of DSB repair enzymes, absence of surveillance factors and mutation or loss of heterozygosity in any of these genes contributes to the pathogenesis of sporadic breast cancers. The results identifying DSB repair defects as a common denominator for breast cancerogenesis focus attention on functional assays in order to assess DSB repair capacity as a diagnostic tool to detect increased breast cancer risk and to enable therapeutic strategies specifically targeting the tumor.

Breast Neoplasms↗

PubMed1998

Repair of radiation-induced DNA double-strand breaks in human fibroblasts is consistent with a continuous spectrum of repair probability.

PURPOSE: To propose a novel interpretation of DNA double-strand break (dsb) repair based on the distribution of energy micro-deposition. MATERIALS AND METHODS: Double-strand break repair curves were studied either after irradiation at 4 degrees C or at 37 degrees C (low dose rate). Two human fibroblast cell lines were used: a control line, HF19, and an ataxia telangiectasia repair-deficient line, AT5BI. Irradiations were made with gamma-rays or alpha-particles (241Am). Repair data were fitted by the variable repair half-time (VRHT) model. Assuming that each dsb has its own inherent repair half-time (IRHT) and that the VRHT is the average of the IRHT at any time during repair, the distribution of the IRHT was calculated. RESULTS: At the end of the irradiation, the distribution was a continuous asymmetric curve with a maximum of dsb having a short IRHT. After 1 h of repair, the curve became bell-shaped. There is a striking similarity between the distribution of dsb repair half-times and that of energy micro-deposition described by Goodhead et al. (1993). CONCLUSION: This similarity suggests a possible causal relationship between the energy density deposition and the repair rate or the probability of dsb repair.

Americium↗

PubMed2006

The loss of gammaH2AX signal is a marker of DNA double strand breaks repair only at low levels of DNA damage.

The induction of DNA double-strand breaks (DSBs) by genotoxic treatment leads to high toxicity and genetic instability. Various approaches have been undertaken to quantify the number of breaks and to follow the kinetic of DSB repair. Recently, the phosphorylation of the variant histone H2AX (named gammaH2AX), quantified by specific immunodetection approaches, has provided a valuable and highly sensitive method to monitor DSBs formation. Although it is admitted that the number of gammaH2AX foci reflected that of DSBs, contradictory reports leave open the question of a link between the disappearance of gammaH2AX signal and DSBs repair. We determined gammaH2AX expression (i) in cells either proficient or not in DSBs repair capacity, (ii) after exposure to ionizing radiation (IR) or calicheamicin gamma1, a radiomimetic compound, (iii) and by three different immunodetection methods, foci numbering, flow cytometry or Western blotting. We showed here that gammaH2AX loss correlates with DSB repair activity only at low cytotoxic doses, when less than 100-150 DSBs breaks per genome are produced, independently of the method used. In addition, in DNA repair proficient cells, the early decrease in the number and intensity of gammaH2AX foci observed after a 2 Gy exposure was not associated with a significant change in the global gammaH2AX level as determined by Western blotting or flow cytometry. These results suggest that the dephosphorylation step of gammaH2AX may be limiting and that the loss of foci is mediated not only by gammaH2AX dephosphorylation but also through its redistribution towards the chromatin.

Aminoglycosides↗

PubMed1996

Measurement of radiation-induced DNA double-strand breaks in human diploid fibroblasts from keloid and normal skin by single-cell gel electrophoresis.

Cell culture experiments recently have shown that different populations of fibroblasts may exist in keloid patients. Moreover, several studies have strongly suggested heterogeneity in cell response of skin fibroblasts in the same individual. The purpose of this study was to evaluate the radiation sensitivity of skin fibroblasts from keloid and normal skin. Single-cell gel electrophoresis was employed in detecting amounts of DNA double-strand breaks generated in individual fibroblasts primarily cultured from normal skin and from keloid tissue after various doses of gamma-irradiation (0, 25, 50, and 100 Gy aerobically) from a 137Cs radiation generator. For the repair study, cells were incubated at 37 degrees C for 0 and 15 minutes and 1 and 2 hours after irradiation. Following neutral lysis and electrophoresis, DNA double-strand breaks were then detected by propidium iodide staining and determined by fluorescence microscope and quantitative image analysis. No significant difference in the extent of DNA double strand breaks in fibroblasts from normal skin or keloid after various doses of irradiation was observed. However, the residual DNA double-strand breaks remaining after various periods of incubation were demonstrated to be significantly increased in fibroblasts from keloid (p < 0.01). In conclusion, different repair capacities were found in fibroblasts from normal skin and keloid scar; this finding may play a role in the treatment of the disease entity.

Cells, Cultured↗

PubMed2025

Arabidopsis thaliana FANCONI ANAEMIA I (FANCI) has roles in the repair of interstrand crosslinks and CRISPR-Cas9 induced DNA double strand breaks.

DNA repair is crucial for genome stability, in particular for plants which are exposed to high levels of damage arising from UV irradiation, soil pollutants and reactive oxygen species. Damage that affects both strands of the DNA duplex is harder to repair due to both the lack of a template strand and the potential for physical separation of fragmented chromosomes. As such, DNA double-strand breaks (DSBs) and interstrand DNA crosslinks (ICL) are particularly cytotoxic forms of damage. Here we report the functions of FANCONI ANAEMIA I (FANCI), an Arabidopsis thaliana homologue of the mammalian ICL repair protein. We show that in plant cells, as in mammals, FANCI forms a nuclear localised complex with FANCD2. Genetic analysis of plants lacking FANCI displays significant hypersensitivity to the DNA crosslinking reagent mitomycin C. Furthermore, mutation of FANCI in combination with mutations in a second ICL repair factor, METHYL METHANESULFONATE AND UV-SENSITIVE PROTEIN 81 (MUS81), results in increased levels of programmed cell death compared to the corresponding single mutants, revealing roles in maintaining plant genome stability. Sequence analysis of mutational repair of CRISPR-Cas9-induced DSBs revealed that FANCI promotes single nucleotide insertions and reduces longer deletions. This pattern of mutations may reflect roles for FA proteins in replication-coupled repair of a subset of DSBs. Taken together, this analysis finds evidence for multiple roles for FANCI in the maintenance of plant genome stability.

Arabidopsis↗

PubMed1986

Repair of DNA double-strand breaks in UV-irradiated Escherichia coli uvrB recF cells is inhibited by rich growth medium.

Ultraviolet (UV)-irradiated uvrB recF and uvrB recB cells of Escherichia coli K-12 showed similar radiation sensitivities when plated on minimal growth medium (MM), however, the uvrB recF cells were much more UV radiation-sensitive than the uvrB recB cells when plated on rich growth medium. Sedimentation analysis of the DNA from UV-irradiated uvrB recF cells suggests that the rich medium killing of uvrB recF cells is due to the inhibition of the repair of UV-radiation-induced DNA double-strand breaks, i.e., the killing is due to the inhibition of the recB-dependent pathway of postreplication repair. Furthermore, we demonstrated that the DNA double-strand breaks that were formed in UV-irradiated uvrB recA200(Ts) cells incubated at 42 degrees C in rich growth medium were not repaired whether the medium during subsequent repair incubation at 30 degrees C was MM or rich growth medium, while DNA double-strand breaks that were formed in MM at 42 degrees C could be repaired in MM or in rich growth medium at 30 degrees C. How the absence of an abrupt slowing of DNA synthesis when UV-irradiated cells are held in rich growth medium (Sharma and Smith, 1985b) may prevent the repair of these DNA double-strand breaks is discussed.

Culture Media↗

PubMed2002

Identification of Ku70 and Ku80 homologues in Arabidopsis thaliana: evidence for a role in the repair of DNA double-strand breaks.

In higher organisms such as mammals and plants, DNA double-strand breaks (DSBs) are repaired preferentially by non-homologous end joining (NHEJ) rather than by homologous recombination. The NHEJ pathway is mediated by Ku, a heterodimer of approximately 70 and 80 kDa subunits, which contributes to various aspects of the metabolism of DNA ends in eukaryotic cells. On the basis of their predicted sequence similarity to human Ku70 and Ku80, cDNAs encoding the first plant homologues of these proteins (AtKu70 and AtKu80, respectively) have now been isolated from Arabidopsis thaliana. AtKu70 and AtKu80 share 28.6 and 22.5% amino acid sequence identity with human Ku70 and Ku80, respectively. Yeast two-hybrid analysis demonstrated that AtKu70 and AtKu80 form a heterodimer, and electrophoretic mobility-shift assays revealed that this heterodimer binds to double-stranded telomeric and non-telomeric DNA sequences, but not to single-stranded DNA. The AtKu heterodimer also possesses single-stranded DNA-dependent ATPase and ATP-dependent DNA helicase activities. Reverse transcription and the polymerase chain reaction revealed that AtKu70 and AtKu80 genes are expressed widely but at low levels in plant tissues. The expression of these two genes in cultured cells was markedly increased in response to the generation of DSBs by bleomycin or methylmethane sulfonate. These results suggest that the evolutionarily conserved Ku70-Ku80 heterodimer functions in DSB repair by the NHEJ pathway in A. thaliana.

Adenosine Triphosphatases↗

PubMed1994

DNA double-strand break repair: genetic determinants of flanking crossing-over.

Whether or not homologous interaction of two DNA molecules results in crossing-over of the flanking sequences is an important decision in view of genome organization. Several homologous recombination models, including the double-strand break repair models, explain this decision as choice between two alternative modes of resolution of Holliday-type intermediates. We have demonstrated that a double-strand gap can be repaired through gene conversion copying a homologous duplex, as predicted by the double-strand break repair models, in the RecE pathway of Escherichia coli. This gap repair is often accompanied by crossing-over of the flanking sequences. Mutations in ruvC and recG, whose products interact with Holliday structures in vitro, do not block double-strand gap repair or its association with flanking crossing-over. However, two mutations in the recJ gene, which encodes a single-strand 5'-->3' exonuclease, severely decrease association of flanking crossing-over. Two mutations in the recQ gene, which encodes a helicase, moderately decrease association of flanking crossing-over by themselves and suppress the severe effect of a recJ mutation. Similar relationships of recJ and recQ mutations are observed in cell survival after ultraviolet light irradiation, gamma-ray irradiation, and H2O2 treatment. We discuss how cooperation of the recQ gene product and the recJ gene product brings about double-strand break repair accompanied by flanking crossing-over. We also discuss how this reaction is related to repair of chromosome damages.

Adenosine Triphosphatases↗

PubMed2001

Severe combined immunodeficient cells expressing mutant hRAD54 exhibit a marked DNA double-strand break repair and error-prone chromosome repair defect.

DNA double-strand breaks (DSBs) can be induced by a number of endogenous and exogenous agents and are lethal events if left unrepaired. DNA DSBs can be repaired by homologous recombination (HR) and nonhomologous end joining (NHEJ). In mammals and higher eukaryotes, NHEJ is thought to be the primary pathway for repair, but the role for each pathway in DNA DSB repair has not been fully elucidated. To define the relative contributions of HR and NHEJ in mammalian DNA DSB repair, cells defective in both pathways were produced. Double-mutant cells were created by expressing a dominant mutant hRAD54 protein in a DNA-dependent protein kinase (DNA-PK)-deficient severe combined immunodeficient cell line. Double-mutant cells demonstrate an increase in ionizing radiation sensitivity and a decrease in DNA DSB repair as compared with either single mutant, whereas single-mutant hRAD54 cells exhibit a wild-type phenotype. Unexpectedly, DNA-PK-null cells were more resistant to mitomycin-C damage than were wild-type cells. Chromosome aberration analysis reveals numerous incomplete chromatid exchange aberrations in the majority of the double-mutant cells after ionizing radiation exposure. Our findings confirm a role for HR in DSB repair in higher eukaryotes, yet indicate that its role is not evident unless the primary repair pathway, NHEJ, is nonfunctional. Mitomycin-C resistance in DNA-PK-null cells compared with wild-type cells suggests that the HR pathway may be more efficient in cross-link repair in the absence of NHEJ. Lastly, the incorrectly repaired chromatid damage observed in double-mutant cells may result from failed recombination or another error-prone repair process that is apparent in the absence of the two primary repair pathways.

Alkylating Agents↗

PubMed1980

Measurement of the kinetics of DNA double strand break repair in Ehrlich ascites tumour cells using the unwinding method.

Two main components of DNA strand break repair have been found using the unwinding method. The first has a time constant (t37) of some minutes and the second, much slower component, a time constant of several hours. The time constant for the slower component of repair was found to vary with the conditions of incubation and to depend on the quality of the radiation. The t37 values obtained for slow repair under various conditions after X-irradiation and after alpha-irradiation were found to be close to those for repair of double strand breaks as measured by velocity sedimentation. Values for initial breaks, obtained by extrapolation of slow repair data back to time zero, were also close to those obtained for double strand breaks. We therefore propose that the unwinding method can be a useful technique for monitoring the repair of double strand breaks.

Alpha Particles↗

PubMed1999

Mouse testicular extracts process DNA double-strand breaks efficiently by DNA end-to-end joining.

DNA double-strand break (DSB) processing was studied in mouse testicular extracts using a defined DSB created by cleaving supercoiled pUC12 DNA at a unique site as the substrate, and analysing the processed DNA by gel electrophoresis. Our results demonstrated that enzymatic activity in the extracts promoted multimerization of DNA and suppressed its circularization. This was distinctly different from T4 DNA ligase activity in the control and therefore the process must be more complex than simple ligation. Efficiency of this end-to-end joining was ATP and Mg(2+)-dependent and was much higher with cohesive (especially with 5') than with blunt ends. On recleaving, the joining was predominantly faithful, especially for cohesive ends; but a detectable fraction of DNA had undergone end-processed joining causing junctional deletions, mostly with blunt ends. Redigestion of end-joined products from time course experiments established that the end-deleted joining occurred concurrent to the faithful joining. Junctional segments were cloned and their restriction analysis confirmed the presence of large deletions from both the sides. These results suggested the association of an end-processing activity (exonuclease/helicase + flap endonuclease) along with the end-joining ligase(s). Suppression of end-edited joining on lowering the reaction temperature to 17 degrees or 14 degrees C, despite efficient faithful joining, indicated that this enzymatic activity is retarded at low temperature. Though the efficiency and fidelity of joining were termini-dependent, the orientation of joining was random. Lack of preference for homologous ends (H:H or T:T), as well as efficient joining of heterologous DNA (pUC12/pBR322) having two different blunt termini, showed that the end joining could occur independent of extensive/terminal homology. Retention of radioactivity on end joining of (alpha-32P)dCTP end-filled cohesive termini, and lack of their junctional cleavability, apparently due to restriction site duplication, suggested direct double strand ligation. Thus it is demonstrated that mouse male germ cells possess an efficient DNA end-joining activity, involving either a major pathway of precise joining, or a minor end-deleted joining, and it seems to be achieved by a multienzymatic complex as suggested also for somatic cells by others. These results show that mammalian male germ cells that are proficient in homologous recombination utilize nonhomologous end-joining (NHEJ) mechanism for DSB processing and therefore NHEJ is a conserved, universal pathway for the vital function of DSB repair.

Adenosine Triphosphate↗

PubMed2005

ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex.

The ataxia-telangiectasia mutated (ATM) kinase signals the presence of DNA double-strand breaks in mammalian cells by phosphorylating proteins that initiate cell-cycle arrest, apoptosis, and DNA repair. We show that the Mre11-Rad50-Nbs1 (MRN) complex acts as a double-strand break sensor for ATM and recruits ATM to broken DNA molecules. Inactive ATM dimers were activated in vitro with DNA in the presence of MRN, leading to phosphorylation of the downstream cellular targets p53 and Chk2. ATM autophosphorylation was not required for monomerization of ATM by MRN. The unwinding of DNA ends by MRN was essential for ATM stimulation, which is consistent with the central role of single-stranded DNA as an evolutionarily conserved signal for DNA damage.

Acid Anhydride Hydrolases↗

PubMed1993

Detection of DNA double-strand breaks through the cell cycle after exposure to X-rays, bleomycin, etoposide and 125IdUrd.

Ionizing radiation-induced DNA double-strand breaks (DSBs) are generally more difficult to detect in S-phase cells than in cells from other phases of the cell cycle. To explore the basis for this observation, other double-strand breaking agents were examined: etoposide, bleomycin and 125IdUrd. DSBs induced by these agents in single cells in S, G1 or G2/M-phases of cell cycle were measured using the neutral comet assay. Regardless of the nature or location of the DSBs, Chinese hamster V79 cells with S-phase DNA content showed about 2-3 times less damage by all agents than cells with G1 or G2/M-phase DNA content. Residual protein content measured after lysis of S-phase cells embedded in agarose did not differ significantly from the protein content of asynchronous cells, and removal of proteins prior to irradiation did not enhance S phase migration. The number of DSBs, the physical nature of the DSB, or the presence of residual proteins, did not appear to influence migration. Therefore, we conclude that differences in DNA structure are responsible for reduced sensitivity for detecting DSBs in S-phase cells.

Animals↗

PubMed1989

Repair of DNA single- and double-strand breaks in proliferating and quiescent murine tumor cells.

We evaluated the relationship between the repair of DNA single- and double-strand breaks and cellular radiosensitivity in proliferating vs. quiescent cells of the mouse mammary tumor lines 66 and 67 in vitro, using the technique of filter elution at pH 12.2, pH 7.2 and pH 9.6. In these lines, quiescent (Q; unfed plateau-phase) cells are more radiosensitive than are proliferating (P) cells. At doses of 4-6 Gy, both 66 and 67 Q cells repair single-strand breaks (ssb) with kinetics similar to those of P cells. However, repair of ssb was slightly retarded in Q cells at a higher dose (10 Gy) than at the lower doses. In contrast, repair of ssb in P cells was dose-independent, at least for doses up to 10 Gy. The rate of repair of DNA double-strand breaks (dsb), measured at pH 7.2, was dose-independent in P and Q cells of both lines. The repair kinetics were biphasic, with an initial half-time less than 15 min, and the early phase was similar in all cell groups. The half-time for repair in the slow phase ranged from about 2 to greater than 20 h. The fraction of damage repaired by the slow phase was relatively high in all cell groups (40-70 per cent). In line 66, P cells repaired a higher percentage of dsb by 2 h postirradiation than did Q cells. The opposite was observed in line 67: Q cells repaired more dsb in 2 h than did P cells. The survival of 66 St4 cells (Q cultures which have been refed with complete medium and incubated 4 h) was significantly greater than that of 66 Q; nevertheless St4 cells repaired both ssb and dsb at rates similar to those of Q cells. Therefore, survival does not necessarily correlate with the rates of either ssb or dsb repair among these cell lines in different growth states.

Animals↗

PubMed2006

Hypothesis: transcript-templated repair of DNA double-strand breaks.

Two mechanisms are available for the repair of DNA double-strand breaks (DSBs) in eukaryotic cells: homology directed repair (HDR) and non-homologous end-joining (NHEJ). While NHEJ is not restricted to a particular phase of the cell cycle, it is incapable of accurately repairing DBSs that have suffered a loss or gain of nucleotide sequence information. In contrast, HDR achieves accurate repair of such DSBs by use of a sister chromatid as a DNA template, but is restricted to cell cycle phases (S/G2) when such templates are available. In this scheme, G1 cells appear to lack a mechanism for the accurate repair of certain DSBs, and an ability to use alternative templates would be highly advantageous. Considered here, therefore, is the possibility that RNA transcripts are used as templates for HDR. Potential mechanisms for transcript-templated HDR, and ways in which it might be detected, are presented.

Animals↗

PubMed1990

Increased sensitivity to killing by restriction enzymes in the XR-1 DNA double-strand break repair-deficient mutant.

Repair or misrepair of DNA double-strand breaks (DSBs) is critical in determining cellular survival after gamma-irradiation. In this report, we focus on the cellular and biochemical consequences of restriction enzyme induced DSBs in wild-type Chinese hamster ovary (CHO) cells and the DNA DSB repair-defective mutant XR-1. We find that XR-1 possesses reduced cellular survival after the introduction of restriction enzymes that produce either cohesive or blunt ends. XR-1's sensitivity to killing by restriction enzymes strongly mimics its response to gamma-rays. Using pulsed field electrophoresis, we find that for each enzyme, similar numbers of DNA DSBs are being introduced in both cell lines. The simplest explanation for the increased sensitivity to restriction enzymes in the mutant is that the biochemical defect in XR-1 is not confined to the repair of ionizing radiation induced ends, but extends to DSBs that possess ligatable 3'-hydroxyl and 5'-phosphate ends as well.

Animals↗

PubMed2005

Enhanced stimulation of chromosomal translocations and sister chromatid exchanges by either HO-induced double-strand breaks or ionizing radiation in Saccharomyces cerevisiae yku70 mutants.

DNA double-strand break (DSB) repair occurs by homologous recombination (HR) or non-homologous endjoining (NHEJ). In Saccharomyces cerevisiae, expression of both MATa and MATalpha inhibits NHEJ and facilitates DSB-initiated HR. We previously observed that DSB-initiated recombination between two his3 fragments, his3-Delta5' and his3-Delta3'::HOcs is enhanced in haploids and diploids expressing both MATa and MATalpha genes, regardless of the position or orientation of the his3 fragments. Herein, we measured frequencies of DNA damage-associated translocations and sister chromatid exchanges (SCEs) in yku70 haploid mutants, defective in NHEJ. Translocation and SCE frequencies were measured in strains containing the same his3 fragments after DSBs were made directly at trp1::his3-Delta3'::HOcs. Wild type and yku70 cells were also exposed to ionizing radiation and radiomimetic agents methyl methanesulfonate (MMS), phleomycin, and 4-nitroquinolone-1-oxide (4-NQO). Frequencies of X-ray-associated and DSB-initiated translocations were five-fold higher in yku70 mutants compared to wild type; however, frequencies of phleomycin-associated translocations were lower in the yku70 haploid mutant. Frequencies of DSB-initiated SCEs were 1.8-fold higher in the yku70 mutant, compared to wild type. Thus, DSB-initiated HR between repeated sequences on non-homologous chromosomes and sister chromatids occurs at higher frequencies in yku70 haploid mutants; however, higher frequencies of DNA damage-associated HR in yku70 mutants depend on the DNA damaging agent.

DNA Damage↗