Unrepaired DNA Double-strand Breaks Research Articles

Senescing human cells and ageing mice accumulate DNA lesions with unrepairable double-strand breaks

Humans and animals undergo ageing, and although their primary cells undergo cellular senescence in culture, the relationship between these two processes is unclear. Here we show that gamma-H2AX foci (gamma-foci), which reveal DNA double-strand breaks (DSBs), accumulate in senescing human cell cultures and in ageing mice. They colocalize with DSB repair factors, but not significantly with telomeres. These cryptogenic gamma-foci remain after repair of radiation-induced gamma-foci, suggesting that they may represent DNA lesions with unrepairable DSBs. Thus, we conclude that accumulation of unrepairable DSBs may have a causal role in mammalian ageing.

DNA-PKcs function regulated specifically by protein phosphatase 5.

Unrepaired DNA double-strand breaks can lead to apoptosis or tumorigenesis. In mammals double-strand breaks are repaired mainly by nonhomologous end-joining mediated by the DNA-PK complex. The core protein of this complex, DNA-PKcs, is a DNA-dependent serine/threonine kinase that phosphorylates protein targets as well as itself. Although the (auto)phosphorylation activity has been shown to be essential for repair of both random double-strand breaks and induced breaks at the immunoglobulin locus, the corresponding phosphatase has been elusive. In fact, to date, none of the putative phosphatases in DNA double-strand break repair has been identified. Here we show that protein phosphatase 5 interacts with DNA-PKcs and dephosphorylates with surprising specificity at least two functional sites. Cells with either hypo- or hyperphosphorylation of DNA-PKcs at these sites show increased radiation sensitivity.

A double-strand break repair defect in ATM-deficient cells contributes to radiosensitivity.

The ATM protein, which is mutated in individuals with ataxia telangiectasia (AT), is central to cell cycle checkpoint responses initiated by DNA double-strand breaks (DSBs). ATM's role in DSB repair is currently unclear as is the basis underlying the radiosensitivity of AT cells. We applied immunofluorescence detection of gamma-H2AX nuclear foci and pulsed-field gel electrophoresis to quantify the repair of DSBs after X-ray doses between 0.02 and 80 Gy in confluence-arrested primary human fibroblasts from normal individuals and patients with mutations in ATM and DNA ligase IV, a core component of the nonhomologous end-joining (NHEJ) repair pathway. Cells with hypomorphic mutations in DNA ligase IV exhibit a substantial repair defect up to 24 h after treatment but continue to repair for several days and finally reach a level of unrepaired DSBs similar to that of wild-type cells. Additionally, the repair defect in NHEJ mutants is dose dependent. ATM-deficient cells, in contrast, repair the majority of DSBs with normal kinetics but fail to repair a subset of breaks, irrespective of the initial number of lesions induced. Significantly, after biologically relevant radiation doses and/or long repair times, the repair defect in AT cells is more pronounced than that of NHEJ mutants and correlates with radiosensitivity. NHEJ-defective cells analyzed for survival following delayed plating after irradiation show substantial recovery while AT cells fail to show any recovery. These data argue that the DSB repair defect underlies a significant component of the radiosensitivity of AT cells.

Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses

DNA double-strand breaks (DSBs) are generally accepted to be the most biologically significant lesion by which ionizing radiation causes cancer and hereditary disease. However, no information on the induction and processing of DSBs after physiologically relevant radiation doses is available. Many of the methods used to measure DSB repair inadvertently introduce this form of damage as part of the methodology, and hence are limited in their sensitivity. Here we present evidence that foci of gamma-H2AX (a phosphorylated histone), detected by immunofluorescence, are quantitatively the same as DSBs and are capable of quantifying the repair of individual DSBs. This finding allows the investigation of DSB repair after radiation doses as low as 1 mGy, an improvement by several orders of magnitude over current methods. Surprisingly, DSBs induced in cultures of nondividing primary human fibroblasts by very low radiation doses (approximately 1 mGy) remain unrepaired for many days, in strong contrast to efficient DSB repair that is observed at higher doses. However, the level of DSBs in irradiated cultures decreases to that of unirradiated cell cultures if the cells are allowed to proliferate after irradiation, and we present evidence that this effect may be caused by an elimination of the cells carrying unrepaired DSBs. The results presented are in contrast to current models of risk assessment that assume that cellular responses are equally efficient at low and high doses, and provide the opportunity to employ gamma-H2AX foci formation as a direct biomarker for human exposure to low quantities of ionizing radiation.

The role of DNA polymerase beta in determining sensitivity to ionizing radiation in human tumor cells.

Lethal lesions after ionizing radiation are thought to be mainly unrepaired or misrepaired DNA double-strand breaks, ultimately leading to lethal chromosome aberrations. However, studies with radioprotectors and repair inhibitors indicate that single-strand breaks, damaged nucleotides or abasic sites can also influence cell survival. This paper reports on studies to further define the role of base damage and base excision repair on the radiosensitivity of human cells. We retrovirally transduced human tumor cells with a dominant negative form of DNA polymerase beta, comprising the 14 kDa DNA-binding domain of DNA polymerase beta but lacking polymerase function. Radiosensitization of two human carcinoma cell lines, A549 and SQD9, was observed, achieving dose enhancement factors of 1.5-1.7. Sensitization was dependent on expression level of the dominant negative and was seen in both single cell clones and in unselected virally transduced populations. Sensitization was not due to changes in cell cycle distribution. Little or no sensitization was seen in G(1)-enriched populations, indicating cell cycle specificity for the observed sensitization. These results contrast with the lack of effect seen in DNA polymerase beta knockout cells, suggesting that polDN also inhibits the long patch, DNA polymerase beta-independent repair pathway. These data demonstrate an important role for BER in determining sensitivity to ionizing radiation and might help identify targets for radiosensitizing tumor cells.

Detecting, signalling and repairing DNA double-strand breaks.

DNA double-strand breaks (DSBs) can be generated by a variety of genotoxic agents, including ionizing radiation and radiomimetic chemicals. They can also occur when DNA replication complexes encounter other forms of DNA damage, and are produced as intermediates during certain site-specific recombination processes. It is crucial that cells recognize DSBs and bring about their efficient repair, because a single unrepaired cellular DSB can induce cell death, and defective DSB repair can lead to mutations or the loss of significant segments of chromosomal material. Eukaryotic cells have evolved a variety of systems to detect DNA DSBs, repair them, and signal their presence to the transcription, cell cycle and apoptotic machineries. In this review, I describe how work on mammalian cells and also on model organisms such as yeasts has revealed that such systems are highly conserved throughout evolution, and has provided insights into the molecular mechanisms by which DNA DSBs are recognized, signalled and repaired. I also explain how defects in the proteins that function in these pathways are associated with a variety of human pathological states.

Detecting, signalling and repairing DNA double-strand breaks

DNA double-strand breaks (DSBs) can be generated by a variety of genotoxic agents, including ionizing radiation and radiomimetic chemicals. They can also occur when DNA replication complexes encounter other forms of DNA damage, and are produced as intermediates during certain site-specific recombination processes. It is crucial that cells recognize DSBs and bring about their efficient repair, because a single unrepaired cellular DSB can induce cell death, and defective DSB repair can lead to mutations or the loss of significant segments of chromosomal material. Eukaryotic cells have evolved a variety of systems to detect DNA DSBs, repair them, and signal their presence to the transcription, cell cycle and apoptotic machineries. In this review, I describe how work on mammalian cells and also on model organisms such as yeasts has revelaed that such systems are highly conserved throughout evolution, and has provided insights into the molecular mechanisms by which DNA DSBs are recognized, signalled and repaired. I also explain how defects in the proteins that function in these pathways are associated with a variety of human pathological states.

SIR functions are required for the toleration of an unrepaired double-strand break in a dispensable yeast chromosome.

Unrepaired DNA double-strand breaks (DSBs) typically result in G(2) arrest. Cell cycle progression can resume following repair of the DSBs or through adaptation to the checkpoint, even if the damage remains unrepaired. We developed a screen for factors in the yeast Saccharomyces cerevisiae that affect checkpoint control and/or viability in response to a single, unrepairable DSB that is induced by HO endonuclease in a dispensable yeast artificial chromosome containing human DNA. SIR2, -3, or -4 mutants exhibit a prolonged, RAD9-dependent G(2) arrest in response to the unrepairable DSB followed by a slow adaptation to the persistent break, leading to division and rearrest in the next G(2). There are a small number of additional cycles before permanent arrest as microcolonies. Thus, SIR genes, which repress silent mating type gene expression, are required for the adaptation and the prevention of indirect lethality resulting from an unrepairable DSB in nonessential DNA. Rapid adaptation to the G(2) checkpoint and high viability were restored in sir(-) strains containing additional deletions of the silent mating type loci HML and HMR, suggesting that genes under mating type control can reduce the toleration of a single DSB. However, coexpression of MATa1 and MATalpha2 in Sir(+) haploid cells did not lead to lethality from the HO-induced DSB, suggesting that toleration of an unrepaired DSB requires more than one Sir(+) function.

Transfer of Ku86 RNA antisense decreases the radioresistance of human fibroblasts.

Ku86 has been shown to be involved in DNA double-strand break (DSB) repair and radiosensitivity in rodents, but its role in human cells is still under investigation. The purpose of this study was to evaluate the radiosensitivity and DSB repair after transfection of a Ku86-antisense in a human fibroblast cell line. Simian virus 40-transformed MRC5V1 human fibroblasts were transfected with a vector (pcDNA3) containing a Ku86-antisense cDNA. The main endpoints were Ku86 protein level, Ku DNA end-binding and DNA protein kinase activity, clonogenic survival, and DSB repair kinetics. After transfection of the Ku86-antisense, decreased Ku86 protein expression, Ku DNA end-binding activity, and DNA protein kinase activity were observed in the uncloned cellular population. The fibroblasts transfected with the Ku86-antisense showed also a radiosensitive phenotype, with a surviving fraction at 2 Gy of 0.29 compared with 0.75 for the control and 20% of unrepaired DSB observed at 24 hours after irradiation compared with 0% for the control. Several clones were also isolated with a decreased level of Ku86 protein, a surviving fraction at 2 Gy between 0.05 and 0.40, and 10-20% of unrepaired DSB at 24 hours. This study is the first to show the implication of Ku86 in DSB repair and in the radiosensitivity of human cells. This investigation strongly suggests that Ku86 could constitute an appealing target for combining gene therapy and radiation therapy.

Underestimation of the small residual damage when measuring DNA double-strand breaks (DSB): is the repair of radiation-induced DSB complete?

Purpose: To overcome the underestimation of the small residual damage when measuring DNA double-strand breaks (DSB) as fraction of activity released (FAR) by pulsed-field gel electrophoresis. Materials and methods: The techniques used to assess DNA damage (e.g. pulsed-field gel electrophoresis, neutral elution, comet assay) do not directly measure the number of DSB. The Blocher model can be used to express data as DSB after irradiation at 4° C by calculating the distribution of all radiation-induced DNA fragments as a function of their size. We have used this model to measure the residual DSB (irradiation at 4° C followed by incubation at 37° C) in untransformed human fibroblasts. Results: The DSB induction rate after irradiation at 4° C was 39.1 +/- 2.0Gy -1. The DSB repair rate obtained after doses of 10 to 80Gy followed by repair times of 0 to 24h was expressed as unrepaired DSB calculated from the Blocher formula. All the damage appeared to be repaired at 24h when the data were expressed as FAR, whereas 15% of DSB remained unrepaired. The DSB repair rate and the chromosome break repair rate assessed by premature condensation chromosome (PCC) techniques were similar. Conclusion: The expression of repair data in terms of FAR dramatically underestimates the amount of unrepaired DNA damage. The Blocher model that takes into account the size distribution of radiation-induced DNA fragments should therefore be used to avoid this bias. Applied to a normal human fibroblast cell line, this model shows that DSB repair is never complete.

Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells.

Eukaryotic cells repair DNA double-strand breaks (DSBs) by at least two pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ). Rad54 participates in the first recombinational repair pathway while Ku proteins are involved in NHEJ. To investigate the distinctive as well as redundant roles of these two repair pathways, we analyzed the mutants RAD54(-/-), KU70(-/-) and RAD54(-/-)/KU70(-/-), generated from the chicken B-cell line DT40. We found that the NHEJ pathway plays a dominant role in repairing gamma-radiation-induced DSBs during G1-early S phase while recombinational repair is preferentially used in late S-G2 phase. RAD54(-/-)/KU70(-/-) cells were profoundly more sensitive to gamma-rays than either single mutant, indicating that the two repair pathways are complementary. Spontaneous chromosomal aberrations and cell death were observed in both RAD54(-/-) and RAD54(-/-)/KU70(-/-) cells, with RAD54(-/-)/KU70(-/-) cells exhibiting significantly higher levels of chromosomal aberrations than RAD54(-/-) cells. These observations provide the first genetic evidence that both repair pathways play a role in maintaining chromosomal DNA during the cell cycle.

Radiation–Induced Chromatid Breaks and DNA Repair in Blood Lymphocytes of Patients with Dysplastic Nevi and/or Cutaneous Melanoma

Each chromatid contains a single continuous molecule of double-stranded DNA, so chromatid breaks represent unrepaired DNA double-strand breaks. Frequencies of chromatid breaks after G2 phase x-irradiation were determined in phytohemagglutinin-stimulated blood lymphocytes from normal subjects and from four categories of patients with dysplastic nevi with or without cutaneous melanoma or with melanoma alone. Some cells were treated with an inhibitor of DNA repair replication to determine enzymatic incision activity at damaged sites after exposure to x-rays, UVC, or fluorescent light. Whereas one of 16 normal controls had deficient DNA repair, all 17 patients from families with hereditary dysplastic nevi with or without melanoma (category I) had a deficiency in repair of radiation-induced DNA damage, manifested as an abnormally high frequency of chromatid breaks after x-irradiation or a reduced capacity to incise the damaged sites after UV exposure. Four of 11 patients with sporadic dysplastic nevi alone (category II) and eight of 12 with sporadic dysplastic nevi and melanoma (category III) showed deficient DNA repair after x-irradiation. One of two patients with sporadic melanoma and no dysplastic nevi (category IV) was also deficient in repair of x-ray-induced damage. Deficient DNA repair thus appears to be associated with hereditary dysplastic nevi with or without melanoma. It also characterizes some patients with sporadic dysplastic nevi or melanoma.

Repair of DNA double-strand breaks and its effect on RBE

DNA double-strand breaks (DSB) are induced linearly with absorbed dose both for sparsely and densely ionizing radiations. By enzymatic repair the linear relationship between the number of DSB and absorbed dose is converted into a non linear one. Furthermore, the RBE-values of high LET radiations for residual DSB increase with increasing amount of DSB repair especially in the low dose range. Unrepaired and/or misrepaired DSB are supposed to be responsible for chromosomal aberrations, cell killing, oncogenic cell transformation and gene mutation. At low doses, for these endpoints much higher RBE-values than those for initial DSB are observed. However, with increasing doses the RBE-values for these endpoints approach those for initial DSB. These observations are likely to be interpreted using the following two parameters of the energy deposition structure: 1. 1. The distribution of clusters with respect to their size at the nm-scale and to the number of ionizations per cluster (cluster distribution). 2. 2. The distribution of distances between clusters of definite size and with definite number of ionizations (distance distribution of clusters). For the induction of DSB solely the ionization density in clusters of nm-dimensions (i. e. the cluster distribution) is important. For unrepaired or misrepaired DSB (responsible for chromosome aberrations, cell killing, oncogenic cell transformation and gene mutation) both the cluster distribution and the distance distribution of clusters are relevant. At low doses the distance distribution of clusters along a single particle track determines the RBE-value. However, with increasing dose the distribution of clusters produced by all particles traversing the cell nucleus becomes increasingly determinant. Here, solely the cluster distribution is important as it is the case for the induction of DSB.

Different fates of camptothecin-induced replication fork-associated double-strand DNA breaks in mammalian cells.

The S phase cytotoxicity of camptothecin (CPT) requires both the formation of a covalent topoisomerase I-DNA complex and ongoing DNA replication. The interaction of DNA synthesis and the drug-induced complexes results in the production of DNA double-strand breaks (DSBs) concentrated in replicating DNA. These DSBs are likely to be extremely cytotoxic lesions and are likely to account for the S phase specificity of CPT. Here we show that a brief exposure to CPT results in replication-associated DSBs and, once formed, the fate of these DNA DSBs is different in human and Chinese hamster cell lines. In hamster CHO-KI, even at supra-lethal concentrations, CPT-induced DSBs in nascent DNA disappear within 5 h of drug removal. Those CHO-KI cells in S phase during treatment with toxic doses of CPT arrive at mitosis within 18 h, with potentially lethal chromatid aberrations. In human cells, CPT-induced DSBs are long lived, and are still detectable at least 24 h after drug removal. After toxic doses of CPT to S phase human cells, mitosis does not occur within 72 h of drug removal and there is an extended, perhaps permanent, cycle arrest in S/G2, possibly due to the presence of unrepaired DNA DSBs. These data, and the greater sensitivity of hamster than human cells to low doses of CPT, suggests that, besides the generation of replication fork-associated DNA DSBs, subsequent processing/repair of these lesions may modulate the sensitivity of cells to this important anti-tumour drug.

Using three-color chromosome painting to test chromosome aberration models.

Ionizing radiation induces DNA double-strand breaks (DSB), which interact pairwise to produce chromosome aberrations. There have long been two main competing theories of such pairwise DSB-DSB interactions. The "classical" theory asserts that an unrepaired DSB makes two ends that separate within the cell nucleus, with each end subsequently able to join any similar (nontelomeric) end. The "exchange" theory asserts that at a DSB the chromatin does not separate completely; rather the DSB ends remain associated until repair, or an illegitimate recombination involving another DSB, occurs. The DSB-DSB interaction mechanism was tested by using three-color fluorescence in situ hybridization to paint chromosomes and observe "three-color triplets": three broken and misrejoined chromosomes having cyclically permuted colors. We observed 18 "three-color triplets" in 2000 cells after 2.25 Gy of gamma-irradiation. On the exchange model in its standard form such three-color triplets cannot occur, so this model is inconsistent with the observations. On the classical model, formalized as a discrete time Markov chain embedded at the transitions of a continuous time Markov chain, the frequency of occurrence of three-color triplets can be computed by Monte Carlo simulations. The number of three-color triplets predicted mathematically by the classical model was found to be slightly larger than the observed number. Thus our data, together with our computer simulations, exclude the standard form of the exchange model but are compatible with the classical model. The results are also compatible with other, more complicated models.

Persistence of radiation-induced double-strand breaks in the DNA of heated CHO cells.

Chinese hamster ovary cells were either heated at 45 degrees C for 15 min or left unheated immediately prior to irradiation and incubation at 37 or 41 degrees C for 5 h. When cellular DNA was analysed by electrophoresis of double-stranded DNA through agarose gels 5 h after irradiation, DNA fragments presumably resulting from unrepaired DNA double-strand breaks (dsbs) were observed in the DNA of all cells. The frequency of the putative unrepaired dsbs was greater in cells heated at 45 degrees C for 15 min before, or incubated at 41 degrees C for 5 h after irradiation, than in unheated, control cells. Gel electrophoresis results were consistent with a failure of irradiated cells to rejoin dsb completely when heated at 45 degrees C before, or incubated at 41 degrees C for 5 h after irradiation. In contrast, nuclear DNA accessibility studies using either an exogenous or an endogenous endonuclease detected no change in the accessibility of DNA in nuclei from 41 degrees C-heated cells. These DNA accessibility studies indicate that the dsbs observed in the DNA of 41 degrees C-heated cells may not result from an actual failure of irradiated cells to repair radiation-induced dsbs during incubation at 41 degrees C.

Cytotoxicity of 125I decay in the DNA double strand break repair deficient mutant cell line, xrs-5.

The survival of parental Chinese hamster ovary (CHO) K1 cells and the DNA double strand break (DSB) repair deficient mutant, xrs-5 was determined after accumulation of 125I decays. Both CHO and xrs-5 cells were extremely sensitive to accumulated 125I decays. The D0 values for CHO and xrs-5 cells were 40 and approximately 7 decays per cell, respectively. The difference in cell survival between CHO and xrs-5 cells was not due to differences in overall 125IUdR incorporation, differences in labelling index (LI) or differences in plating efficiency (PE). Relative biological effectiveness (RBE) values calculated relative to 137Cs gamma radiation survival values (D0 and D10) were higher in xrs-5 cells compared with CHO cells. Although both CHO and xrs-5 cells have high RBE values that correspond to a high sensitivity of CHO and xrs-5 cells to 125I decay. The higher RBE observed for xrs-5 cells in combination with the known repair defect in xrs-5 cells support the idea that unrepaired DNA double strand breaks are lethal to the cell.

Effects of 3'-deoxyadenosine (cordycepin) on the repair of X-ray-induced DNA single- and double-strand breaks in Chinese hamster V79 cells.

The ability of cordycepin to inhibit the repair of DNA strand breaks was examined with X-irradiated Chinese hamster V79 cells in log-phase culture. A filter elution technique revealed that 70 microM cordycepin did not inhibit the repair of single-strand breaks but inhibited the repair of double-strand breaks. These findings confirmed the fact that the increase in the lethality of cordycepin in X-irradiated cultured mammalian cells was attributable to unrepaired DNA double-strand breaks.

A Model Relating Cell Survival to DNA Fragment Loss and Unrepaired Double-Strand Breaks

The model of radiation action that is presented relates the surviving fraction of irradiated cells to unrepaired DNA double-strand breaks (DSBs). The following assumptions are made in the model: (i) A DNA fragment created by the induced DSBs may move out of its chromosome (become lost), and the probability of that process depends on the fragment size. (ii) An irradiated cell will lose its proliferative capacity if it has an unrepaired DSB (including DNA fragments) at certain points in the cell cycle. Mathematical expressions of the model yield the dose and time dependencies of the surviving fraction, the number of unrepaired DSBs, and the number of prematurely condensed chromosome fragments. Radiobiological phenomena described include effects of low dose rate, delayed plating, hypertonic solution, araA, and high-LET radiation. The calculated dose dependence of the residual number of unrepaired DSBs for ataxia telangiectasia and normal fibroblast cells is very close to the experimentally obtained [M. N. Cornforth and J. S. Bedford, Radiat. Res. 111, 385-405 (1987)] total number of chromosomal aberrations. This leads to the conclusion that each unrepaired DSB becomes a chromosomal aberration. Analysis in terms of the model shows that the radiosensitivity of various cell lines is predominantly due to the different amounts of time available for DSB repair in these cells.

Neoplastic transformation of human cells in culture associated with deficient repair of light‐induced chromosomal DNA damage

AbstractTwo lines of normal human skin fibroblasts and five derivative lines transformed in culture to neoplastic cells by chemical carcinogens were compared with respect to chromatid breakage produced by exposure to low‐intensity fluorescent light (cool‐white, 4.6 W/m2) during pre‐S, S, and late S‐G2 periods of the cell cycle. Five additional normal human cell lines and a line derived from a lung adenocarcinoma were also examined for chromatid damage following light exposure during pre‐S and late S‐G2. On the assumption that each chromatid (half chromosome) contains a single continuous DNA double helix, chromatid breaks seen in the first post‐treatment metaphase must represent unrepaired DNA doublestrand breaks. The frequency of chromatid breaks induced by pre‐S light exposure in the neoplastic cell is not significantly different from that in the normal cells. However, addition of caffeine, an inhibitor of DNA repair, to the culture medium following light exposure significantly increases the frequency of chromatid breaks in the neoplastic but not in the normal cells. One possible explanation for these observations is that the normal cells, known to have effective excision repair, remove the DNA damage, whereas the neoplastic cells, presumably deficient in excision repair, handle the damage during S by a caffeinesensitive mechanism. Furthermore, the neoplastic but not the normal cells, show significant increases in chromatid breaks when exposed during S or late S‐G2The chromatid damage produced by late S‐G2 exposure can be completely prevented by addition of mannitol, a scavenger of free hydroxyl radicals. Addition of caffeine to the culture medium during late S‐G2 light exposure has no effect on the frequency of chromatid breaks in the neoplastic cells but signincantly increases chromatid breakage in the normal cells. These results suggest that the normal cells repair the late S‐G2 light‐induced damage by a caffeine‐sensitive repair mechanism that is absent or deficient in the neoplastic cells. It thus appears that neoplastic cells are deficient in mechanisms for repair of DNA damage induced by light both during pre‐S and late S‐G2periods of the cell cycle.