Rejoining Of DNA Double-strand Breaks Research Articles

O-GlcNAcylation Affects the Pathway Choice of DNA Double-Strand Break Repair.

Exposing cells to DNA damaging agents, such as ionizing radiation (IR) or cytotoxic chemicals, can cause DNA double-strand breaks (DSBs), which are crucial to repair to maintain genetic integrity. O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a post-translational modification (PTM), which has been reported to be involved in the DNA damage response (DDR) and chromatin remodeling. Here, we investigated the impact of O-GlcNAcylation on the DDR, DSB repair and chromatin status in more detail. We also applied charged particle irradiation to analyze differences of O-GlcNAcylation and its impact on DSB repair in respect of spatial dose deposition and radiation quality. Various techniques were used, such as the γH2AX foci assay, live cell microscopy and Fluorescence Lifetime Microscopy (FLIM) to detect DSB rejoining, protein accumulation and chromatin states after treating the cells with O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) inhibitors. We confirmed that O-GlcNAcylation of MDC1 is increased upon irradiation and identified additional repair factors related to Homologous Recombination (HR), CtIP and BRCA1, which were increasingly O-GlcNAcyated upon irradiation. This is consistent with our findings that the function of HR is affected by OGT inhibition. Besides, we found that OGT and OGA activity modulate chromatin compaction states, providing a potential additional level of DNA-repair regulation.

Ku70 suppresses alternative end joining in G1-arrested progenitor B cells

Classical nonhomologous end joining (C-NHEJ) repairs DNA double-strand breaks (DSBs) throughout interphase but predominates in G1 phase when homologous recombination is unavailable. Complexes containing the Ku70/80 ("Ku") and XRCC4/ligase IV (Lig4) core C-NHEJ factors are required, respectively, for sensing and joining DSBs. While XRCC4/Lig4 are absolutely required for joining RAG1/2 endonuclease ("RAG")-initiated DSBs during V(D)J recombination in G1-phase progenitor lymphocytes, cycling cells deficient for XRCC4/Lig4 also can join chromosomal DSBs by alternative end-joining (A-EJ) pathways. Restriction of V(D)J recombination by XRCC4/Lig4-mediated joining has been attributed to RAG shepherding V(D)J DSBs exclusively into the C-NHEJ pathway. Here, we report that A-EJ of DSB ends generated by RAG1/2, Cas9:gRNA, and Zinc finger endonucleases in Lig4-deficient G1-arrested progenitor B cell lines is suppressed by Ku. Thus, while diverse DSBs remain largely as free broken ends in Lig4-deficient G1-arrested progenitor B cells, deletion of Ku70 increases DSB rejoining and translocation levels to those observed in Ku70-deficient counterparts. Correspondingly, while RAG-initiated V(D)J DSB joining is abrogated in Lig4-deficient G1-arrested progenitor B cell lines, joining of RAG-generated DSBs in Ku70-deficient and Ku70/Lig4 double-deficient lines occurs through a translocation-like A-EJ mechanism. Thus, in G1-arrested, Lig4-deficient progenitor B cells are functionally end-joining suppressed due to Ku-dependent blockage of A-EJ, potentially in association with G1-phase down-regulation of Lig1. Finally, we suggest that differential impacts of Ku deficiency versus Lig4 deficiency on V(D)J recombination, neuronal apoptosis, and embryonic development results from Ku-mediated inhibition of A-EJ in the G1 cell cycle phase in Lig4-deficient developing lymphocyte and neuronal cells.

Roles for the DNA-PK complex and 53BP1 in protecting ends from resection during DNA double-strand break repair.

p53-binding protein 1 (53BP1) exerts distinct impacts in different situations involving DNA double-strand break (DSB) rejoining. Here we focus on how 53BP1 impacts upon the repair of ionising radiation-induced DSBs (IR-DSBs) and how it interfaces with Ku, the DNA end-binding component of canonical non-homologous end-joining (c-NHEJ), the major DSB repair pathway in mammalian cells. We delineate three forms of IR-DSB repair: resection-independent c-NHEJ, which rejoins most IR-DSBs with fast kinetics in G1 and G2, and Artemis and resection-dependent c-NHEJ and homologous recombination (HR), which repair IR-DSBs with slow kinetics in G1 and G2 phase, respectively. The fast component of DSB repair after X-ray exposure occurs via c-NHEJ with normal kinetics in the absence of 53BP1. Ku is highly abundant and has avid DNA end-binding capacity which restricts DNA end-resection and promotes resection-independent c-NHEJ at most IR-DSBs. Thus, 53BP1 is largely dispensable for resection-independent c-NHEJ. In contrast, 53BP1 is essential for the process of rejoining IR-DSBs with slow kinetics. This role requires 53BP1’s breast cancer susceptibility gene I (BRCA1) C-terminal (BRCT) 2 domain, persistent ataxia telangiectasia mutated (ATM) activation and potentially relaxation of compacted chromatin at heterochromatic-DSBs. In distinction, 53BP1 inhibits resection-dependent IR-DSB repair in G1 and G2, and this resection-inhibitory function can be counteracted by BRCA1. We discuss a model whereby most IR-DSBs are rapidly repaired by 53BP1-independent and resection-independent c-NHEJ due to the ability of Ku to inhibit resection, but, if delayed, then resection in the presence of Ku is triggered, the 53BP1 barrier comes into force and BRCA1 counteraction is required for resection.

Premature chromosome condensation assay to study influence of high-level natural radiation on the initial DNA double strand break repair in human G0 lymphocytes

The inherent capacity of individuals to efficiently repair ionizing radiation induced DNA double strand breaks (DSBs) may be inherited, however, it is influenced by several epigenetic and environmental factors. A pilot study tested whether chronic low dose natural radiation exposure influences the rejoining of initial DNA DSBs induced by a 2 Gy γ-irradiation in 22 individuals from high (>1.5 mGy/year) and normal (≤1.5 mGy/year) level natural radiation areas (H&NLNRA) of Kerala. Rejoining of DSBs (during 1 h at 37 °C, immediately after irradiation) was evaluated at the chromosome level in the presence and absence of wortmannin (a potent inhibitor of DSB repair in normal human cells) using a cell fusion-induced premature chromosome condensation (PCC) assay. The PCC assay quantitates DSBs in the form of excess chromosome fragments in human G0 lymphocytes without the requirement for cell division. A quantitative difference was observed in the early rejoining of DNA DSBs between individuals from HLNRA and NLNRA, with HLNRA individuals showing a higher (P = 0.05) mean initial repair ratio. The results indicate an influence of chronic low dose natural radiation on initial DNA DSB repair in inhabitants of HLNRA of the Kerala coast.

Tumor treating fields cause replication stress and interfere with DNA replication fork maintenance: Implications for cancer therapy

Tumor treating fields (TTFields) is a noninvasive physical modality of cancer therapy that applies low-intensity, intermediate frequency, and alternating electric fields to a tumor. Interference with mitosis was the first mechanism describing the effects of TTFields on cancer cells; however, TTFields was shown to not only reduce the rejoining of radiation-induced DNA double-strand breaks (DSBs), but to also induce DNA DSBs. The mechanism(s) by which TTFields generates DNA DSBs is related to the generation of replication stress including reduced expression of the DNA replication complex genes MCM6 and MCM10 and the Fanconi's Anemia pathway genes. When markers of DNA replication stress as a result of TTFields exposure were examined, newly replicated DNA length was reduced with TTFields exposure time and there was increased R-loop formation. Furthermore, as cells were exposed to TTFields a conditional vulnerability environment developed which rendered cells more susceptible to DNA damaging agents or agents that interfere with DNA repair or replication fork maintenance. The effect of TTFields exposure with concomitant exposure to cisplatin or PARP inhibition, the combination of TTFields plus concomitant PARP inhibition followed by radiation, or radiation alone at the end of a TTFields exposure were all synergistic. Finally, gene expression analysis of 47 key mitosis regulator genes suggested that TTFields-induced mitotic aberrations and DNA damage/replication stress events, although intimately linked to one another, are likely initiated independently of one another. This suggests that enhanced replication stress and reduced DNA repair capacity are also major mechanisms of TTFields effects, effects for which there are therapeutic implications.

Enhancing low-dose risk assessment using mechanistic mathematical models of radiation effects

Mechanistic mathematical modeling of ionizing radiation (IR) effects has a long history spanning several decades. Models that mathematically represent current knowledge and hypotheses about how radiation damages cells and organs, leading to deleterious outcomes such as carcinogenesis, are particularly useful for estimating radiation risks at doses that are relevant for radiation protection, but are too low to provide a strong ‘signal-to-noise ratio’ in epidemiological or experimental studies with realistic sample sizes. Here, I discuss examples of models in several relevant areas, including radionuclide biokinetics, non-targeted IR effects, DNA double-strand break (DSB) rejoining and radiation carcinogenesis. I do not provide a detailed review of the vast modeling literature in these fields, but focus on concepts that we have implemented, such as using continuous probability distributions of exponential rates to model radionuclide biokinetics and DSB rejoining, and combining short and long time scales in carcinogenesis models. Improvements in models, including the ability to generate new hypotheses based on model predictions, may come from the introduction of additional novel concepts and from integrating multiple data types.

Induction and rejoining of DNA double-strand breaks in the lymphocytes of prostate cancer patients after radium-223 treatment as assessed by the γH2AX foci assay.

The aim of this study is to assess if the number of radiation-induced double strand breaks (DSB) in lymphocytes of prostate cancer patients is affected after repeated Ra-223 therapies. In addition, we investigated the repair of ex vivo induced DSB to investigate the repair proficiency in patient's lymphocytes over the therapy course. Before each of six therapy cycles, blood samples were obtained from seventeen patients. After separation of lymphocytes, the cells were subjected to immunofluorescence staining for detection of DSB-marking γH2AX foci. The number of foci per cell per patient sample was determined for each cycle (X1-X6, baseline foci per cell). Additionally, appropriate samples were exposed ex vivo to an X-ray dose of 1 Gy. The number of γH2AX foci per cell were analyzed after 0.5 h, 2 h and 24 h of recovery. Patient-specific linear regression of the baseline foci per cell over the therapy cycles revealed no significant slopes in the regression lines. Likewise, the mean baseline foci per cell of all patients for cycles X2-X6 was not significantly elevated in comparison to the pre-therapeutic value (X1). The differences between the percentages of residual DSB and cycles were not significant, both at 2 h and 24 h repair time. Consideration of the X6/X1 ratios of both the number of lymphocytes and the amount of residual damage at 24 h indicated a significant correlation. Our findings indicate that the number of γH2AX foci per cell was not changed in dependence on the Ra-223 therapy cycles. The ability of patient's lymphocytes to repair ex vivo induced DSB remained unaffected throughout the entire therapy course.

DNA-dependent protein kinase: effect on DSB repair, G2/M checkpoint and mode of cell death in NSCLC cell lines

Purpose: To evaluate the effect of NU7026, a specific inhibitor of DNA-PKcs, on DNA-double strand break (DSB) repair in a cell cycle specific manner, on the G2/M checkpoint, mitotic progression, apoptosis and clonogenic survival in non-small-cell lung carcinoma (NSCLC) cell lines with different p53 status.Material and methods: Cell cycle progression, and hyperploidy were evaluated using flow cytometry. Polynucleation as a measure for mitotic catastrophe (MC) was evaluated by fluorescence microscopy. DSB induction and repair were measured by constant-gel electrophoresis and γH2AX assay. The efficiency of DSB rejoining during the cell cycle was assessed by distinguishing G1 and G2/M phase cells on the basis of the DNA content in flow cytometry. The overall effect on cell death was determined by apoptosis and the surviving fraction after irradiation with 2 Gy (SF2) assessed by clonogenic survival.Results: DSB signaling upon treatment with NU7026, as measured by γH2AX signaling, was differently affected in G1 and G2/M cells. The background level of γH2AX was significantly higher in G2/M compared to G1 cells, whereas NU7026 had no effect on the background level. The steepness of the initial dose effect relation at 1 h after irradiation was less pronounced in G2/M compared to G1 cells. NU7026 had no significant effect on the initial dose-effect relation of γH2AX signaling. In comparison, NU7026 significantly slowed down the repair kinetics and increased the residual γH2AX signal at 24 h after irradiation in the G1 phase of all cell lines, but was less effective in G2/M cells. NU7026 significantly increased the fraction of G2/M phase cells upon irradiation. Moreover, NU7026 significantly increased mitotic catastrophe and hyperploidy, as a measure for mitotic failure after low irradiation doses of about 4 Gy, but decreased both at higher doses of 20 Gy. In addition, radiation induced apoptosis increased in A549, H520 and H460 but decreased in H661 upon NU7026 treatment, with a significant reduction of SF2 in all NSCLC cell lines.Conclusion: Overall, NU7026 significantly influences the cell cycle progression through the G2- and M-phases and thereby determines the fate of cells. The impairment of DNA-PK upon treatment with NU7026 affects the efficiency of the NHEJ system in a cell cycle dependent manner, which may be of relevance for a clinical application of DNA-PK inhibitors in tumor therapy.

The G-quadruplex-stabilizing ligand RHPS4 enhances sensitivity of U251MG glioblastoma cells to clinical carbon ion beams.

The pentacyclic acridine RHPS4 is a highly potent and specific G-quadruplex (G4) ligand, which binds and stabilizes telomeric G4 leading to the block of the replication forks at telomeres and consequently to telomere dysfunctionalization. In turn, the cell recognizes unprotected telomeres as DNA double-strand breaks with consequent activation of DNA repair response at telomeres, cellular growth impairment, and death. Data from the literature showed the capability of this compound to sensitize U251MG glioblastoma radioresistant cell line to X-rays sparsely ionizing radiations. In the present paper, it was investigated whether RHPS4 is also able to increase the effect of clinical carbon ion beams (cells irradiated in the middle of a spread-out Bragg peak, in the energy range of 246-312MeV·μm-1 and a dose-averaged linear energy transfer of 46keV·μm-1 ). Interestingly, also for charged particles whose damage inflicted to DNA is more complex than that of sparsely ionizing radiations and results in higher Relative Biological Effectiveness (RBE), RHPS4 significantly potentiated the radiation effect in terms of cell killing, delayed rejoining of DNA double-strand breaks (γ-H2AX and 53BBP1 immunofluorescence staining), chromosome aberrations (pan-centromeric/telomeric FISH and multicolor FISH), and G2 /M-phase accumulation in GBM cells. Overall, the results provide the first evidence that the combined administration of the G4-ligand RHPS4 with charged particles interfere with cellular processes involved in cell survival leading to radiosensitization of highly radioresistant tumor cells.

DNA-damage response in hematopoietic stem cells: an evolutionary trade-off between blood regeneration and leukemia suppression.

Self-renewing and multipotent hematopoietic stem cells (HSCs) maintain lifelong hematopoiesis. Their enormous regenerative potential coupled with lifetime persistence in the body, in contrast with the Progenitors, demand tight control of HSCs genome stability. Indeed, failure to accurately repair DNA damage in HSCs is associated with bone marrow failure and accelerated leukemogenesis. Recent observations exposed remarkable differences in several DNA-damage response (DDR) aspects between HSCs and Progenitors, especially in their DNA-repair capacities and susceptibility to apoptosis. Human HSCs in comparison with Progenitors exhibit delayed DNA double-strand break rejoining, persistent DDR signaling activation, higher sensitivity to the cytotoxic effects of ionizing radiation and attenuated expression of DNA-repair genes. Importantly, the distinct DDR of HSCs was also documented in mouse models. Nevertheless, physiological significance and the molecular basis of the HSCs-specific DDR features are only partially understood. Taking radiation-induced DDR as a paradigm, this review will focus on the current advances in understanding the role of cell-intrinsic DDR regulators and the cellular microenvironment in balancing stemness with genome stability. Pre-leukemia HSCs and clonal hematopoiesis evolvement will be discussed as an evolutionary compromise between the need for lifelong blood regeneration and DDR. Uniquely for this review, we outline the differences in HSCs-related DDR as highlighted by various experimental systems and attempt to provide their critical analysis.

Influence of reduced glutathione on end-joining of DNA double-strand breaks: Cytogenetical and molecular approach

Radiation induced DNA double-strand breaks (DSB) are the major initial lesions whose misrejoining may lead to exchange aberrations. However, the role of glutathione (GSH), a major cellular thiol, in regulating cell’s sensitivity to DNA damaging agents is not well understood. Influence of endogenous GSH on the efficiency of X-rays and bleomycin (Blem) induced DNA DSBs end-joining has been tested here cytogenetically, in human lymphocytes and Hct116 cells. In another approach, oligomeric DNA (75bp) containing 5′-compatible and non-compatible overhangs mimicking the endogenous DSB were for rejoining in presence of cell-free extracts from cells having different endogenous GSH levels. Frequency of aberrations, particularly exchange aberrations, was significantly increased when Blem was combined with radiation. The exchange aberration frequency was further enhanced when combined treatment was given at 4°C since DNA lesions are poorly repaired at 4°C so that a higher number of DNA breaks persist and interact when shifted from 4°C to 37°C. The exchange aberrations increased further when the combined treatment was given to Glutathione-ester (GE) pre-treated cells, indicating more frequent rejoining of DNA lesions in presence of higher cellular GSH. This is further supported by the drastic reduction in frequency of exchange aberrations but significant increase in incidences of deletions when combined treatment was given to GSH-depleted cells. End-joining efficiency of DNA DSBs with compatible ends was better than for non-compatible ends. End-joining efficiency of testicular and MCF7 cell extracts was better than that of lungs and Hct116 cells. Cell extract made from GE-treated MCF-7 cells provided more efficient end-joining than from untreated and GSH-depleted cells. However, direct addition of GSH to the cell-free extracts showed considerable reduction in end-joining efficiency. Present data indicate that higher endogenous GSH favours rejoining of DNA DSBs (both restitution and illegitimate reunion) which in turn produce more exchange aberrations.

Efficient Rejoining of DNA Double-Strand Breaks despite Increased Cell-Killing Effectiveness following Spread-Out Bragg Peak Carbon-Ion Irradiation.

Radiotherapy of solid tumors with charged particles holds several advantages in comparison to photon therapy; among them conformal dose distribution in the tumor, improved sparing of tumor-surrounding healthy tissue, and an increased relative biological effectiveness (RBE) in the tumor target volume in the case of ions heavier than protons. A crucial factor of the biological effects is DNA damage, of which DNA double-strand breaks (DSBs) are the most deleterious. The reparability of these lesions determines the cell survival after irradiation and thus the RBE. Interestingly, using phosphorylated H2AX as a DSB marker, our data in human fibroblasts revealed that after therapy-relevant spread-out Bragg peak irradiation with carbon ions DSBs are very efficiently rejoined, despite an increased RBE for cell survival. This suggests that misrepair plays an important role in the increased RBE of heavy-ion radiation. Possible sources of erroneous repair will be discussed.

Mechanistic Modeling of Dose and Dose Rate Dependences of Radiation-Induced DNA Double Strand Break Rejoining Kinetics in Saccharomyces cerevisiae.

Mechanistic modeling of DNA double strand break (DSB) rejoining is important for quantifying and medically exploiting radiation-induced cytotoxicity (e.g. in cancer radiotherapy). Most radiation-induced DSBs are quickly-rejoinable and are rejoined within the first 1–2 hours after irradiation. Others are slowly-rejoinable (persist for several hours), and yet others are essentially unrejoinable (persist for >24 hours). The dependences of DSB rejoining kinetics on radiation dose and dose rate remain incompletely understood. We hypothesize that the fraction of slowly-rejoinable and/or unrejoinable DSBs increases with increasing dose/dose rate. This radiation-dependent (RD) model was implemented using differential equations for three DSB classes: quickly-rejoinable, slowly-rejoinable and unrejoinable. Radiation converts quickly-rejoinable to slowly-rejoinable, and slowly-rejoinable to unrejoinable DSBs. We used large published data sets on DSB rejoining in yeast exposed to sparsely-ionizing (electrons and γ-rays, single or split-doses, high or low dose rates) and densely-ionizing (α-particles) radiation to compare the performances of the proposed RD formalism and the established two-lesion kinetic (TLK) model. These yeast DSB rejoining data were measured within the radiation dose range relevant for clonogenic cell survival, whereas in mammalian cells DSB rejoining is usually measured only at supra-lethal doses for technical reasons. The RD model described both sparsely-ionizing and densely-ionizing radiation data much better than the TLK model: by 217 and 14 sample-size-adjusted Akaike information criterion units, respectively. This occurred because: the RD (but not the TLK) model reproduced the observed upwardly-curving dose responses for slowly-rejoinable/unrejoinable DSBs at long times after irradiation; the RD model adequately described DSB yields at both high and low dose rates using one parameter set, whereas the TLK model overestimated low dose rate data. These results support the hypothesis that DSB rejoining is progressively impeded at increasing radiation doses/dose rates.

New Insight into Quantitative Modeling of DNA Double-Strand Break Rejoining.

Accurate and mechanistically plausible mathematical models of DNA double-strand break (DSB) rejoining kinetics are needed to correctly estimate the dependence of cell death and transformation on linear energy transfer, radiation dose and time. When integrated into more comprehensive risk estimation approaches, such models are potentially valuable tools in applications such as treatment planning for radiotherapy. In this study, we compared 10 DSB rejoining models based on data collected from 61 mammalian cell lines after high-dose-rate photon or heavy ion irradiation. The set of models included formalisms with: 1. one, two or three discrete first-order rejoining rates; 2. continuously distributed first-order rejoining rates; and 3. second-order rejoining rates. The Akaike information criterion was used to quantify the relative support for each model from the data, accounting for goodness of fit and model complexity. The best performance was exhibited by a bi-exponential model with two discrete rejoining rates and a model with gamma-distribution rejoining rates. Models with more than three free parameters overfitted the data and models with single DSB rejoining rates or with an inflexible distribution of rejoining rates lacked accuracy. Of special note is that the analyzed data provide little support for models that rely on pairwise interactions to describe DSB rejoining kinetics. Consequently, kinetic cell survival models reflecting bi-exponential DSB rejoining might be preferable to models based on the kinetics of intra- and inter-lesion rejoining.

Low doses of X-rays induce prolonged and ATM-independent persistence of γH2AX foci in human gingival mesenchymal stem cells.

Diagnostic imaging delivering low doses of radiation often accompany human mesenchymal stem cells (MSCs)-based therapies. However, effects of low dose radiation on MSCs are poorly characterized. Here we examine patterns of phosphorylated histone H2AX (γH2AX) and phospho-S1981 ATM (pATM) foci formation in human gingiva-derived MSCs exposed to X-rays in time-course and dose-response experiments. Both γH2AX and pATM foci accumulated linearly with dose early after irradiation (5-60 min), with a maximum induction observed at 30-60 min (37 ± 3 and 32 ± 3 foci/cell/Gy for γH2AX and pATM, respectively). The number of γH2AX foci produced by intermediate doses (160 and 250 mGy) significantly decreased (40-60%) between 60 and 240 min post-irradiation, indicating rejoining of DNA double-strand breaks. In contrast, γH2AX foci produced by low doses (20-80 mGy) did not change after 60 min. The number of pATM foci between 60 and 240 min decreased down to control values in a dose-independent manner. Similar kinetics was observed for pATM foci co-localized with γH2AX foci. Collectively, our results suggest differential DNA double-strand break signaling and processing in response to low vs. intermediate doses of X-rays in human MSCs. Furthermore, mechanisms governing the prolonged persistence of γH2AX foci in these cells appear to be ATM-independent.

Application of the local effect model to predict DNA double-strand break rejoining after photon and high-LET irradiation.

In the recent version of the local effect model (LEM), the biological effects of ionising radiation can be well described trough the consideration of DNA double-strand breaks (DSB) clustering at the micrometre scale. Assuming a giant-loop organisation for the chromatin higher-order structure, two classes of DSB are defined, namely isolated (iDSB) and clustered DSB (cDSB), according to whether exactly one or more than one DSB are induced in a loop, respectively. Here, a DSB kinetic rejoining model based on the LEM is applied to the description of two specific aspects of DSB rejoining, namely the dose dependence of the rejoining capacity after photon radiation and the residual damage observed at late times after ion irradiation. Based on the hypothesis that iDSB and cDSB can be associated to the fast and slow components of rejoining, the model is able to reproduce the experimental data, therefore supporting the relevance of micrometre scale clustering of damage for photon radiation as well as for high-LET radiation.

Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair

Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80–95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure which is uncoupled from its essential function in DSB repair. This could have implications for the development of therapeutic strategies aiming to radiosensitize tumors by affecting the DNA-PKcs function.

MuLV-related endogenous retroviral elements and Flt3 participate in aberrant end-joining events that promote B-cell leukemogenesis.

During V(D)J recombination of immunoglobulin genes, p53 and nonhomologous end-joining (NHEJ) suppress aberrant rejoining of DNA double-strand breaks induced by recombinase-activating genes (Rags)-1/2, thus maintaining genomic stability and limiting malignant transformation during B-cell development. However, Rag deficiency does not prevent B-cell leukemogenesis in p53/NHEJ mutant mice, revealing that p53 and NHEJ also suppress Rag-independent mechanisms of B-cell leukemogenesis. Using several cytogenomic approaches, we identified a novel class of activating mutations in Fms-like tyrosine kinase 3 (Flt3), a receptor tyrosine kinase important for normal hematopoiesis in Rag/p53/NHEJ triple-mutant (TM) B-cell leukemias. These mutant Flt3 alleles were created by complex genomic rearrangements with Moloney leukemia virus (MuLV)-related endogenous retroviral (ERV) elements, generating ERV-Flt3 fusion genes encoding an N-terminally truncated mutant form of Flt3 (trFlt3) that was transcribed from ERV long terminal repeats. trFlt3 protein lacked most of the Flt3 extracellular domain and induced ligand-independent STAT5 phosphorylation and proliferation of hematopoietic progenitor cells. Furthermore, expression of trFlt3 in p53/NHEJ mutant hematopoietic progenitor cells promoted development of clinically aggressive B-cell leukemia. Thus, repetitive MuLV-related ERV sequences can participate in aberrant end-joining events that promote development of aggressive B-cell leukemia.

Modeling Damage Complexity-Dependent Non-Homologous End-Joining Repair Pathway

Non-homologous end joining (NHEJ) is the dominant DNA double strand break (DSB) repair pathway and involves several repair proteins such as Ku, DNA-PKcs, and XRCC4. It has been experimentally shown that the choice of NHEJ proteins is determined by the complexity of DSB. In this paper, we built a mathematical model, based on published data, to study how NHEJ depends on the damage complexity. Under an appropriate set of parameters obtained by minimization technique, we can simulate the kinetics of foci track formation in fluorescently tagged mammalian cells, Ku80-EGFP and DNA-PKcs-YFP for simple and complex DSB repair, respectively, in good agreement with the published experimental data, supporting the notion that simple DSB undergo fast repair in a Ku-dependent, DNA-PKcs-independent manner, while complex DSB repair requires additional DNA-PKcs for end processing, resulting in its slow repair, additionally resulting in slower release rate of Ku and the joining rate of complex DNA ends. Based on the numerous experimental descriptions, we investigated several models to describe the kinetics for complex DSB repair. An important prediction of our model is that the rejoining of complex DSBs is through a process of synapsis formation, similar to a second order reaction between ends, rather than first order break filling/joining. The synapsis formation (SF) model allows for diffusion of ends before the synapsis formation, which is precluded in the first order model by the rapid coupling of ends. Therefore, the SF model also predicts the higher number of chromosomal aberrations observed with high linear energy transfer (LET) radiation due to the higher proportion of complex DSBs compared to low LET radiation, and an increased probability of misrejoin following diffusion before the synapsis is formed, while the first order model does not provide a mechanism for the increased effectiveness in chromosomal aberrations observed.

Misrepair of DNA double-strand breaks in patient with unidentified chromosomal fragility syndrome and family history of radiosensitivity

Purpose: To test the hypothesis that differences in DNA double-strand breaks (DSB) repair fidelity underlies differences in radiosensitivity.Materials and methods: A primary fibroblast culture (C42) derived from a pediatric cancer patient treated with reduced radiation doses consequent to a family history of radiosensitivity reminiscent of chromosomal fragility syndrome, was compared to a normal control (C29). DNA DSB rejoining and repair fidelity were studied by Southern blotting and hybridization to specific fragments: Alu repetitive sequence representing the overall DSB rejoining capacity in the genome and a 3.2 Mbp NotI restriction fragment on chromosome 21 for DSB repair fidelity.Results: Although both assays showed statistically significant difference (p ≤ 0.05) between the two cell strains in residual misrepaired (un-or mis-rejoined) DSB (24 h after 30 or 80 Gy), the residual damage was lower in the Alu enriched genome assay compared to NotI assay (0.01–0.07 and 0.10–0.37, respectively).Conclusions: These results suggest that, in comparison to classic DSB repair experiment, an assay of measuring DNA DSB repair fidelity can provide better resolution and a more accurate estimate of misrepair of radiation-induced DNA damage, which underlies genomic instability and increased radiosensitivity.