Complex DNA Double-strand Breaks Research Articles

Biological Consequences of Radiation-induced DNA Damage: Relevance to Radiotherapy

DNA damage of exposed tumour tissue leading to cell death is one of the detrimental effects of ionising radiation that is exploited, with beneficial consequences, for radiotherapy. The pattern of the discrete energy depositions during passage of the ionising track of radiation defines the spatial distribution of lesions induced in DNA with a fraction of the DNA damage sites containing clusters of lesions, formed over a few nanometres, against a background of endogenously induced individual lesions. These clustered DNA damage sites, which may be considered as a signature of ionising radiation, underlie the deleterious biological consequences of ionising radiation. The concepts developed rely in part on the fact that ionising radiation creates significant levels of clustered DNA damage, including complex double-strand breaks (DSB), to kill tumour cells as clustered damage sites are difficult to repair. This reduced repairability of clustered DNA damage using specific repair pathways is exploitable in radiotherapy for the treatment of cancer. We discuss some potential strategies to enhance radiosensitivity by targeting the repair pathways of radiation-induced clustered damage and complex DNA DSB, through inhibition of specific proteins that are not required in the repair pathways for endogenous damage. The variety and severity of DNA damage from ionising radiation is also influenced by the tumour microenvironment, being especially sensitive to the oxygen status of the cells. For instance, nitric oxide is known to influence the types of damage induced by radiation under hypoxic conditions. A potential strategy based on bioreductive activation of pro-drugs to release nitric oxide is discussed as an approach to deliver nitric oxide to hypoxic tumours during radiotherapy. The ultimate aim of this review is to stimulate thinking on how knowledge of the complexity of radiation-induced DNA damage may contribute to the development of adjuncts to radiotherapy.

The dynamics of Ku70/80 and DNA-PKcs at DSBs induced by ionizing radiation is dependent on the complexity of damage

DNA double-strand breaks (DSBs) are biologically one of the most important cellular lesions and possess varying degrees of chemical complexity. The notion that the repairability of more chemically complex DSBs is inefficient led to the concept that the extent of DSB complexity underlies the severity of the biological consequences. The repair of DSBs by non-homologous end joining (NHEJ) has been extensively studied but it remains unknown whether more complex DSBs require a different sub-set of NHEJ protein for their repair compared with simple DSBs. To address this, we have induced DSBs in fluorescently tagged mammalian cells (Ku80-EGFP, DNA-PKcs-YFP or XRCC4-GFP, key proteins in NHEJ) using ultra-soft X-rays (USX) or multi-photon near infrared (NIR) laser irradiation. We have shown in real-time that simple DSBs, induced by USX or NIR microbeam irradiation, are repaired rapidly involving Ku70/80 and XRCC4/Ligase IV/XLF. In contrast, DSBs with greater chemical complexity are repaired slowly involving not only Ku70/80 and XRCC4/Ligase IV/XLF but also DNA-PKcs. Ataxia telangiectasia-mutated inhibition only retards repair of the more chemically complex DSBs which require DNA-PKcs. In summary, the repair of DSBs by NHEJ is highly regulated with pathway choice and kinetics of repair dependent on the chemical complexity of the DSB.

Differences in Phosphorylated Histone H2AX Foci Formation and Removal of Cells Exposed to Low and High Linear Energy Transfer Radiation

The use of particle ion beams in cancer radiotherapy has a long history. Today, beams of protons or heavy ions, predominantly carbon ions, can be accelerated to precisely calculated energies which can be accurately targeted to tumors. This particle therapy works by damaging the DNA of tissue cells, ultimately causing their death. Among the different types of DNA lesions, the formation of DNA double strand breaks is considered to be the most relevant of deleterious damages of ionizing radiation in cells. It is well-known that the extremely large localized energy deposition can lead to complex types of DNA double strand breaks. These effects can lead to cell death, mutations, genomic instability, or carcinogenesis. Complex double strand breaks can increase the probability of mis-rejoining by NHEJ. As a consequence differences in the repair kinetics following high and low LET irradiation qualities are attributed mainly to quantitative differences in their contributions of the fast and slow repair component. In general, there is a higher contribution of the slow component of DNA double strand repair after exposure to high LET radiation, which is thought to reflect the increased amount of complex DNA double strand breaks. These can be accurately measured by the γ-H2AX assay, because the number of phosphorylated H2AX foci correlates well with the number of double strand breaks induced by low or / and high LET radiation.

Irreparable complex DNA double-strand breaks induce chromosome breakage in organotypic three-dimensional human lung epithelial cell culture

DNA damage and consequent mutations initiate the multistep carcinogenic process. Differentiated cells have a reduced capacity to repair DNA lesions, but the biological impact of unrepaired DNA lesions in differentiated lung epithelial cells is unclear. Here, we used a novel organotypic human lung three-dimensional (3D) model to investigate the biological significance of unrepaired DNA lesions in differentiated lung epithelial cells. We showed, consistent with existing notions that the kinetics of loss of simple double-strand breaks (DSBs) were significantly reduced in organotypic 3D culture compared to kinetics of repair in two-dimensional (2D) culture. Strikingly, we found that, unlike simple DSBs, a majority of complex DNA lesions were irreparable in organotypic 3D culture. Levels of expression of multiple DNA damage repair pathway genes were significantly reduced in the organotypic 3D culture compared with those in 2D culture providing molecular evidence for the defective DNA damage repair in organotypic culture. Further, when differentiated cells with unrepaired DNA lesions re-entered the cell cycle, they manifested a spectrum of gross-chromosomal aberrations in mitosis. Our data suggest that downregulation of multiple DNA repair pathway genes in differentiated cells renders them vulnerable to DSBs, promoting genome instability that may lead to carcinogenesis.

Factors determining DNA double-strand break repair pathway choice in G2 phase.

DNA non-homologous end joining (NHEJ) and homologous recombination (HR) function to repair DNA double-strand breaks (DSBs) in G2 phase with HR preferentially repairing heterochromatin-associated DSBs (HC-DSBs). Here, we examine the regulation of repair pathway usage at two-ended DSBs in G2. We identify the speed of DSB repair as a major component influencing repair pathway usage showing that DNA damage and chromatin complexity are factors influencing DSB repair rate and pathway choice. Loss of NHEJ proteins also slows DSB repair allowing increased resection. However, expression of an autophosphorylation-defective DNA-PKcs mutant, which binds DSBs but precludes the completion of NHEJ, dramatically reduces DSB end resection at all DSBs. In contrast, loss of HR does not impair repair by NHEJ although CtIP-dependent end resection precludes NHEJ usage. We propose that NHEJ initially attempts to repair DSBs and, if rapid rejoining does not ensue, then resection occurs promoting repair by HR. Finally, we identify novel roles for ATM in regulating DSB end resection; an indirect role in promoting KAP-1-dependent chromatin relaxation and a direct role in phosphorylating and activating CtIP.

Loss of p15/Ink4b accompanies tumorigenesis triggered by complex DNA double-strand breaks

DNA double-strand breaks (DSBs) are the most deleterious lesion inflicted by ionizing radiation. Although DSBs are potentially carcinogenic, it is not clear whether complex DSBs that are refractory to repair are more potently tumorigenic compared with simple breaks that can be rapidly repaired, correctly or incorrectly, by mammalian cells. We previously demonstrated that complex DSBs induced by high-linear energy transfer (LET) Fe ions are repaired slowly and incompletely, whereas those induced by low-LET gamma rays are repaired efficiently by mammalian cells. To determine whether Fe-induced DSBs are more potently tumorigenic than gamma ray-induced breaks, we irradiated 'sensitized' murine astrocytes that were deficient in Ink4a and Arf tumor suppressors and injected the surviving cells subcutaneously into nude mice. Using this model system, we find that Fe ions are potently tumorigenic, generating tumors with significantly higher frequency and shorter latency compared with tumors generated by gamma rays. Tumor formation by Fe-irradiated cells is accompanied by rampant genomic instability and multiple genomic changes, the most interesting of which is loss of the p15/Ink4b tumor suppressor due to deletion of a chromosomal region harboring the CDKN2A and CDKN2B loci. The additional loss of p15/Ink4b in tumors derived from cells that are already deficient in p16/Ink4a bolsters the hypothesis that p15 plays an important role in tumor suppression, especially in the absence of p16. Indeed, we find that reexpression of p15 in tumor-derived cells significantly attenuates the tumorigenic potential of these cells, indicating that p15 loss may be a critical event in tumorigenesis triggered by complex DSBs.

Dynamic Dependence on ATR and ATM for Double-Strand Break Repair in Human Embryonic Stem Cells and Neural Descendants

The DNA double-strand break (DSB) is the most toxic form of DNA damage. Studies aimed at characterizing DNA repair during development suggest that homologous recombination repair (HRR) is more critical in pluripotent cells compared to differentiated somatic cells in which nonhomologous end joining (NHEJ) is dominant. We have characterized the DNA damage response (DDR) and quality of DNA double-strand break (DSB) repair in human embryonic stem cells (hESCs), and in vitro-derived neural cells. Resolution of ionizing radiation-induced foci (IRIF) was used as a surrogate for DSB repair. The resolution of γ-H2AX foci occurred at a slower rate in hESCs compared to neural progenitors (NPs) and astrocytes perhaps reflective of more complex DSB repair in hESCs. In addition, the resolution of RAD51 foci, indicative of active homologous recombination repair (HRR), showed that hESCs as well as NPs have high capacity for HRR, whereas astrocytes do not. Importantly, the ATM kinase was shown to be critical for foci formation in astrocytes, but not in hESCs, suggesting that the DDR is different in these cells. Blocking the ATM kinase in astrocytes not only prevented the formation but also completely disassembled preformed repair foci. The ability of hESCs to form IRIF was abrogated with caffeine and siRNAs targeted against ATR, implicating that hESCs rely on ATR, rather than ATM for regulating DSB repair. This relationship dynamically changed as cells differentiated. Interestingly, while the inhibition of the DNA-PKcs kinase (and presumably non-homologous endjoining [NHEJ]) in astrocytes slowed IRIF resolution it did not in hESCs, suggesting that repair in hESCs does not utilize DNA-PKcs. Altogether, our results show that hESCs have efficient DSB repair that is largely ATR-dependent HRR, whereas astrocytes critically depend on ATM for NHEJ, which, in part, is DNA-PKcs-independent.

Rejoining of gamma-ray-induced DNA damage in Cryptosporidium parvum measured by the comet assay

Cryptosporidium parvum is a well-known waterborne intracellular protozoan that causes severe diarrheal illness in immunocompromised individuals. This organism is highly resistant to harsh environmental conditions and various disinfectants, and it exhibits one of the highest known resistances to gamma irradiation. We investigated rejoining of gamma-ray-induced DNA damage in C. parvum by neutral comet assay. Oocysts were gamma irradiated at various doses (1, 5, 10, and 25kGy) and were incubated for various periods (6–96h) after exposure to 10kGy. The comet tail moment showed that the number of DNA double-strand breaks increased concomitantly with the gamma irradiation dose. When investigating rejoining after irradiation at 10kGy, double-strand breaks peaked at 6h postirradiation, and rejoining was highest at 72h postirradiation. The observed rejoining pattern suggests that repair process occurs slowly even when complex DNA double-strand breaks in C. parvum were induced by high dose irradiation, 10kGy.

Research Highlights

Research Highlights

MERIT40 controls BRCA1–Rap80 complex integrity and recruitment to DNA double-strand breaks

Rap80 targets the breast cancer suppressor protein BRCA1 along with Abraxas and the BRCC36 deubiquitinating enzyme (DUB) to polyubiquitin structures at DNA double-strand breaks (DSBs). These DSB targeting events are essential for BRCA1-dependent DNA damage response-induced checkpoint and repair functions. Here, we identify MERIT40 (Mediator of Rap80 Interactions and Targeting 40 kD)/(C19orf62) as a Rap80-associated protein that is essential for BRCA1-Rap80 complex protein interactions, stability, and DSB targeting. Moreover, MERIT40 is required for Rap80-associated lysine(63)-ubiquitin DUB activity, a critical component of BRCA1-Rap80 G2 checkpoint and viability responses to ionizing radiation. Thus, MERIT40 represents a novel factor that links BRCA1-Rap80 complex integrity, DSB recognition, and ubiquitin chain hydrolytic activities to the DNA damage response. These findings provide new molecular insights into how BRCA1 associates with independently assembled core protein complexes to maintain genome integrity.

Single-Strand Annealing, Conservative Homologous Recombination, Nonhomologous DNA End Joining, and the Cell Cycle-Dependent Repair of DNA Double-Strand Breaks Induced by Sparsely or Densely Ionizing Radiation

The cell cycle-dependent relative contributions of error-prone single-strand annealing (SSA), error-free conservative homologous recombination (HR), and potentially error-prone nonhomologous DNA end joining (NHEJ) to repair simple (induced by 200 kV X rays) or complex (induced by (241)Am alpha particles) DNA double-strand breaks (DSBs) in Chinese hamster ovary cells are reported for the first time. Cells of the parental cell line AA8 and its derivatives UV41 (SSA-deficient), irs1SF (HR-deficient) and V3 (NHEJ-deficient) were synchronized in G(1) or in S phase, and survival responses after exposure to either type of radiation were measured. It is demonstrated for the first time that in G(1)-phase SSA is negligible for the repair of DSBs of various complexities. HR-deficient cells exposed to X rays or alpha particles in G(1) phase show enhanced radiosensitivity, but this does not necessarily mean that HR is important in G(1) phase. NHEJ appears to be the most important (if not the only) mechanism in G(1) phase acting efficiently on simple DSBs, but complex DSBs are a less preferred target. In contrast to X rays, NHEJ-deficient cells show no cell cycle-dependent variation in sensitivity to alpha particles. Surprisingly, when these cells are exposed to X rays in G(1) phase, they are even more sensitive compared to alpha particles. It is also shown for the first time that in S phase all three mechanisms play a role in the repair of simple and complex DSBs. A defect in SSA confers radiosensitivity to cells in S phase, suggesting that the error-prone SSA mechanism is important for the repair of specific simple and complex DSBs that are not a substrate for HR or NHEJ. The most important mechanism in S phase for the repair of simple and complex DSBs is HR. This is also emphasized by the finding that irs1SF cells, after complementation of their HR defect by human XRCC3 cDNA, show a greater radioresistance than parental cells, whereas resistance to mitomycin C is only partially restored. Complementation confers a greater resistance to alpha particles than X rays, suggesting an important role of HR, especially for the repair of complex DSBs. In S phase, NHEJ is more important than SSA for the repair of simple DSBs, but SSA is more important than NHEJ for the repair of complex DSBs.

The Role of Nonhomologous DNA End Joining, Conservative Homologous Recombination, and Single-Strand Annealing in the Cell Cycle-Dependent Repair of DNA Double-Strand Breaks Induced by H2O2in Mammalian Cells

The purpose of this study was to investigate the cell cycle-dependent role of nonhomologous DNA end joining (NHEJ), conservative homologous recombination (HR), and single-strand annealing (SSA) for the repair of simple DNA double-strand breaks (DSBs) induced by H(2)O(2)-mediated OH radicals in CHO cells. Cells of the cell lines V3 (NHEJ-deficient), irs1SF (HR-deficient) and UV41 (SSA-deficient) and their parental cell line AA8 were exposed to various concentrations of H(2)O(2) in G(1) or S phase of the cell cycle and their colony-forming ability was assayed. In G(1) phase, NHEJ was the most important-if not the only-mechanism to repair H(2)O(2)-mediated DSBs; this was similar to results obtained in a parallel study of more complex DSBs induced by sparsely or densely ionizing radiation. Unlike HR (irs1SF)- and SSA (UV41)-deficient cells, the sensitivity of NHEJ-deficient V3 cells to H(2)O(2) relative to parental AA8 cells in G(1) phase is about 50 times higher compared to 200 kV X rays. This points to a specific role of the catalytic subunit of DNA-PK for efficient NHEJ of H(2)O(2)-mediated DSBs that are located at sites critical for the maintenance of the higher-order structure of cellular DNA, whereas X-ray-induced DSBs are distributed stochastically. Surprisingly, SSA-deficient cells in G(1) phase showed an increased sensitivity to high concentrations of H(2)O(2) relative to the parental wild-type cells and to HR-deficient cells, which may be interpreted in terms of a specific type of H(2)O(2)-induced damage requiring SSA for repair after its transfer into S phase. In S phase, HR is the most important mechanism to repair H(2)O(2)-mediated DSBs, followed by NHEJ. In contrast, the action of error-prone SSA may not be beneficial, since SSA-deficient cells are three times more resistant to H(2)O(2) than wild-type AA8 cells. This is likely due to channeling of DSBs into the error-free HR repair pathway or into the potentially error-prone NHEJ pathway. Cells with or without a defect in DSB repair are considerably more sensitive to H(2)O(2) in S phase compared to G(1) phase. This effect is likely due to the fact that topoisomerase II, which is expressed only in proliferating cells, is a target of H(2)O(2), resulting in enhanced accumulation of DSBs and killing of cells treated in S phase with H(2)O(2). The relative sensitivities to H(2)O(2) differ by orders of magnitude for the four cell lines. This seems to be caused mainly by H(2)O(2)-mediated poisoning of topoisomerase IIalpha rather than by a defect in DSB repair.

ATM Signaling Facilitates Repair of DNA Double-Strand Breaks Associated with Heterochromatin

Ataxia Telangiectasia Mutated (ATM) signaling is essential for the repair of a subset of DNA double-strand breaks (DSBs); however, its precise role is unclear. Here, we show that < or =25% of DSBs require ATM signaling for repair, and this percentage correlates with increased chromatin but not damage complexity. Importantly, we demonstrate that heterochromatic DSBs are generally repaired more slowly than euchromatic DSBs, and ATM signaling is specifically required for DSB repair within heterochromatin. Significantly, knockdown of the transcriptional repressor KAP-1, an ATM substrate, or the heterochromatin-building factors HP1 or HDAC1/2 alleviates the requirement for ATM in DSB repair. We propose that ATM signaling temporarily perturbs heterochromatin via KAP-1, which is critical for DSB repair/processing within otherwise compacted/inflexible chromatin. In support of this, ATM signaling alters KAP-1 affinity for chromatin enriched for heterochromatic factors. These data suggest that the importance of ATM signaling for DSB repair increases as the heterochromatic component of a genome expands.

SU-FF-T-360: Probabilistic Two-Stage Model of Cell Inactivation by Ionizing Radiation: The Model, Its Interpretation, and Relation to Kinetic Radiobiological Models

Purpose: To discuss the recently proposed probabilistic model of cell inactivation, possible biophysical interpretation of the involved damage classification, and the relation of the model to kinetic radiobiological models. Method and Materials: The probabilistic two stage model takes into account two classes of DNA damage of different severity. While even a single unrepaired lethal lesion is assumed to inactivate the cell, less severe lesions have to combine in at least pairwise manner. Repair success probabilities, strongly varying between different cell lines, are represented. Results: The probabilistic model enables to represent in a systematic manner a large variety of survival curves observed experimentally, including also the low-dose hypersensitivity phenomena. Under some conditions, the probabilistic model can be viewed as a solution to kinetic models of radiation action, such as the lethal-potentially lethal, repair-misrepair, or saturable repair model. E.g. for acute irradiation and fully developed endpoints, assuming non-saturated repair processes, the model predicts survival curves with a linear and a logarithmic term, similarly to the predictions of kinetic models, and can be approximated in terms of the linear-quadratic model, too. To address possible biophysical interpretation of the damage classification used in the probabilistic model, a detailed analysis of published survival data for V79 cells irradiated by low-energy protons was performed. Comparison with the yields of complex DNA lesions, estimated by Monte Carlo methods, indicates that lethal lesions correlate with a specific subset of complex DNA double-strand breaks. Conclusion: The probabilistic model is a valuable tool for analyzing and interpreting radiobiological data. It enables damage induction and repair be described systematically. Development of a detailed microscopic radiobiological model is desirable to estimate damage probabilities in dependence on radiation quality on a model basis and thus increase the predictive power of the present model scheme.

Low levels of clustered oxidative DNA damage induced at low and high LET irradiation in mammalian cells

DNA double-strand breaks (DSBs) and locally multiply damaged sites (LMDS) induced by ionizing radiation (IR) are considered to be very genotoxic in mammalian cells. LMDS consist of two or more clustered DNA lesions including oxidative damage locally formed within one or two helical turns by single radiation tracks following local energy deposition. They are thought to be frequently induced by IR but not by normal oxidative metabolism. In mammalian cells, LMDS are detected after specific enzymatic treatments transforming these lesions into additional DSBs that can be revealed by pulsed-field gel electrophoresis (PFGE). Here, we studied radiation-induced DSBs and LMDS in Chinese hamster ovary cells (CHO-K1). After addition of the iron chelator deferoxamine (DFO) or the antioxidant glutathione (GSH) to the cell lysis solution, we observed reduced spontaneous DNA fragmentation and a clear dose-dependent increase of radiation-induced DSBs. LMDS induction, however, was close to background levels, independently of dose, dose rate, temperature and radiation quality (low and high LET). Under these experimental conditions, artefactual oxidative DNA damage during cell lysis could not anymore be confounded with LMDS. We thus show that radiation-induced LMDS composed of oxidized purines or pyrimidines are much less frequent than hitherto reported, and suggest that they may be of minor importance in the radiation response than DSBs. We speculate that complex DSBs with oxidized ends may constitute the main part of radiation-induced clustered lesions. However, this needs further studies.

Detailed analysis of the cell-inactivation mechanism by accelerated protons and light ions

A detailed study of the biological effects of diverse quality radiations, addressing their biophysical interpretation, is presented. Published survival data for V79 cells irradiated by monoenergetic protons, helium-3, carbon and oxygen ions and for CHO cells irradiated by carbon ions have been analysed using the probabilistic two-stage model of cell inactivation. Three different classes of DNA damage formed by traversing particles have been distinguished, namely severe single-track lesions which might lead to cell inactivation directly, less severe lesions where cell inactivation is caused by their combinations and lesions of negligible severity that can be repaired easily. Probabilities of single ions forming these lesions have been assessed in dependence on their linear energy transfer (LET) values. Damage induction probabilities increase with atomic number and LET. While combined lesions play a crucial role at lower LET values, single-track damage dominates in high-LET regions. The yields of single-track lethal lesions for protons have been compared with Monte Carlo estimates of complex DNA lesions, indicating that lethal events correlate well with complex DNA double-strand breaks. The decrease in the single-track damage probability for protons of LET above approximately 30 keV µm−1, suggested by limited experimental evidence, is discussed, together with the consequent differences in the mechanisms of biological effects between protons and heavier ions. Applications of the results in hadrontherapy treatment planning are outlined.

Low-Dose Binary Behavior of Bystander Cell Killing after Microbeam Irradiation of a Single Cell with Focused CKX Rays

Although conclusive evidence has been obtained for the presence of radiation-induced bystander effects, the mechanisms that trigger and regulate these processes are still largely unknown. The bystander effect may play a critical role in determining the biological effectiveness of low-dose exposures, but questions on how to incorporate it into current models and extrapolate the risks of radiation-induced carcinogenesis are still open. The Gray Cancer Institute soft X-ray microbeam has been used to investigate the dose-response relationship of the bystander effect below 0.5 Gy. The survival response of V79 cells was assessed after the irradiation of a single cell within a population with a submicrometer-size beam of carbon K X rays (278 eV). Above 0.3 Gy, the measured bystander cell killing was in agreement with previously published data; however, a significant increase in the scatter of the data was observed in the low-dose region (<0.3 Gy). The data distribution observed indicates a binary behavior for triggering of the bystander response. According to our hypothesis, the probability of triggering a bystander response increases approximately linearly with the dose delivered to the single selected cell, reaching 100% above about 0.3 Gy. The magnitude of the bystander effect, when triggered, is approximately constant with the dose and results in an overall approximately 10% reduction in survival in our system. This suggests that the event that triggers the emission of the bystander signal by the hit cell is an all-or-nothing process. Extrapolation of the data indicates that when a single fast electron traverses a V79 cell, there is a probability of approximately 0.3% that the cell will emit the bystander signal. The data presented in this paper have also been analyzed statistically to test the possibility that complex DNA double-strand breaks may be the initial critical event.

Repair of a minimal DNA double-strand break by NHEJ requires DNA-PKcs and is controlled by the ATM/ATR checkpoint

Mammalian cells primarily rejoin DNA double-strand breaks (DSBs) by the non-homologous end-joining (NHEJ) pathway. The joining of the broken DNA ends appears directly without template and accuracy is ensured by the NHEJ factors that are under ATM/ATR regulated checkpoint control. In the current study we report the engineering of a mono-specific DNA damaging agent. This was used to study the molecular requirements for the repair of the least complex DSB in vivo. Single-chain PvuII restriction enzymes fused to protein delivery sequences transduce cells efficiently and induce blunt end DSBs in vivo. We demonstrate that beside XRCC4/LigaseIV and KU, the DNA-PK catalytic subunit (DNA-PKcs) is also essential for the joining of this low complex DSB in vivo. The appearance of blunt end 3'-hydroxyl and 5'-phosphate DNA DSBs induces a significantly higher frequency of anaphase bridges in cells that do not contain functional DNA-PKcs, suggesting an absolute requirement for DNA-PKcs in the control of chromosomal stability during end joining. Moreover, these minimal blunt end DSBs are sufficient to induce a p53 and ATM/ATR checkpoint function.

Induction of chromatid breaks by carbon K-shell ultrasoft X rays.

Chromatid breaks have previously been shown to be induced in G2-phase cells after exposure to ionizing radiation (X and gamma rays) as a linear function of dose, consistent with a single-event mechanism. DNA double-strand breaks (DSBs) are thought to be the initiating lesion, and experiments with a genetically engineered cell line containing a single DSB site also indicate that a single DSB is sufficient to induce a chromatid break. Although the precise mechanism of conversion of an isolated DSB into a chromatid break is not yet understood, it is known that a proportion of chromatid breaks result from rearrangements between sister chromatids. Here we report further evidence for the single-event hypothesis for the formation of chromatid breaks. The evidence derives from experiments in which chromatid breaks have been induced by exposure of Chinese hamster cells to ultrasoft carbon K-shell X rays. Since the energy of carbon K-shell X rays is not sufficient for the secondary electrons to span more than one DNA double helix, we conclude that single traversals, and hence single (complex) DSBs, are responsible for the formation of chromatid breaks. We find that, as for 60Co gamma rays, around 10% of the carbon K-shell X-ray-induced chromatid breaks have associated color switches at breakpoints, indicating that they arise through sister chromatid rearrangements.

Model for the initiation of ionizing radiation-induced apoptosis in lymphoid cells by complex DNA double-strand breaks

Purpose : To present a model for the molecular events that lead to the induction of apoptosis in irradiated lymphoid cells based on the assumption that the process is triggered by complex DNA double-strand breaks (DSB). Outline of the model : •Cellular DNA repair mechanisms have difficulty rejoining complex DSB because of the nature of the end groups on such breaks. •Association between p53 and DNA topoisomerase I (topo I) can occur at complex DSB in open regions of the genome and the enzymic activity of such associations is not suppressed by polyADP-ribosylation. •Binding of p53 and topo I at a complex DSB results in the transient trapping of a DNA-topo I cleavage complex. •Transiently trapped DNA-topo I cleavage complexes at complex DSB are reversed following association with topo I bound elsewhere in the genome, thus initiating a misrejoining event. •Topo I-mediated DNA misrejoining creates a structure that activates p53. Initiation of rapid interphase apoptosis requires that the inducing signal from activated p53 exceeds a threshold level. •Initiation of rapid interphase apoptosis is regulated by poly(ADP-ribose) polymerase.