Misrepair Of DNA Double-strand Breaks Research Articles

Abstract 7107: Clustered DNA double strand breaks for cancer treatment via multi-gRNA CRISPR system

Abstract Radiation therapy is one of the most common cancer treatments. Ionizing radiation generates DNA double-strand breaks (DSBs), which are the most lethal type of DNA damage. The dominant pathway for repairing DSBs is non-homologous end joining (NHEJ), but homologous recombination (HR) is also important. NHEJ is dominant pathway in all cell cycle phases while HR is largely restricted to S and G2 phases. Unrepaired or misrepaired DSBs can lead to cell death. KU70/80 and DNA-PKcs are essential for NHEJ. KU70/80 binds to broken ends, and DNA-PKcs binds to DNA-bound KU70/80, which activates DNA-PKs kinase allowing NHEJ to proceed. Clustered DSBs (two or more DSBs separated by <200 bp) generate small DNA fragments that are efficiently bound by KU70/80 and DNA-PKcs, but fail to activate DNA-PKcs kinase, inhibiting NHEJ and shifting repair toward HR. Thus, clustered DSBs are poorly repaired and more cytotoxic than dispersed DSBs. There are two types of ionization radiation: high LET and low LET. High LET radiation has a higher ionization density and therefore induces more clustered DSBs than low LET, which explains why high LET radiation is more cytotoxic at a given dose. A major limitation of radiotherapy is normal tissue damage. We hypothesize that mimicking high-LET-induced clustered DSBs using CRISPR/Cas9 targeted to cancer-specific DNA sequences (e.g., indel mutations) will effectively kill cancer cells but spare normal cells. In this approach, DSBs are not targeted to specific genes, nor is CRISPR/Cas9 used for gene editing. Instead, cancer cell killing is achieved by inducing poorly repaired DSBs anywhere in cancer cell DNA. Thus, this therapeutic approach is distinct from conventional targeted therapies that are oncogenic pathway-dependent, and this eliminates oncogenic pathway-dependent resistance mechanisms. However, types of resistance are possible, such as upregulated DSB repair. Our data indicate that the cytotoxicity of clustered DSBs depends on the number of small DNA fragments induced, thus we observe similar cytotoxicity with a localized, 4-DSB cluster and with three non-localized 2-DSB clusters, as each generates 3 small fragments per target chromosome. This finding significantly increases available targets as 2-DSB clusters can be induced at indels and flanking (wild-type) sites, generating clustered DSBs in cancer cells but efficiently repaired dispersed DSBs in normal cells lacking the cancer indel. Data also indicates that a single DSB targeted to each amplicon of highly amplified regions is also cytotoxic, and we are exploring the effectiveness of combination clustered DSBs targeted to amplified regions. We are also exploring how DSB repair inhibitors (like ATMi, DNA-PKcsi or RAD51i) can augment cytotoxic DSB effects, potentially mitigating that resistance pathway. These proof-of-principal studies represent the first approach to targeted cancer therapy that is oncogenic pathway-independent. Citation Format: Gamze Badakul, Neelam Sharma, Jac A. Nickoloff. Clustered DNA double strand breaks for cancer treatment via multi-gRNA CRISPR system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7107.

MicroRNA-145 Impairs Classical Non-Homologous End-Joining in Response to Ionizing Radiation-Induced DNA Double-Strand Breaks via Targeting DNA-PKcs.

DNA double-strand breaks (DSBs) are one of the most lethal types of DNA damage due to the fact that unrepaired or mis-repaired DSBs lead to genomic instability or chromosomal aberrations, thereby causing cell death or tumorigenesis. The classical non-homologous end-joining pathway (c-NHEJ) is the major repair mechanism for rejoining DSBs, and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a critical factor in this pathway; however, regulation of DNA-PKcs expression remains unknown. In this study, we demonstrate that miR-145 directly suppresses DNA-PKcs by binding to the 3′-UTR and inhibiting translation, thereby causing an accumulation of DNA damage, impairing c-NHEJ, and rendering cells hypersensitive to ionizing radiation (IR). Of note, miR-145-mediated suppression of DNA damage repair and enhanced IR sensitivity were both reversed by either inhibiting miR-145 or overexpressing DNA-PKcs. In addition, we show that the levels of Akt1 phosphorylation in cancer cells are correlated with miR-145 suppression and DNA-PKcs upregulation. Furthermore, the overexpression of miR-145 in Akt1-suppressed cells inhibited c-NHEJ by downregulating DNA-PKcs. These results reveal a novel miRNA-mediated regulation of DNA repair and identify miR-145 as an important regulator of c-NHEJ.

Boon and Bane of DNA Double-Strand Breaks.

DNA double-strand breaks (DSBs), interrupting the genetic information, are elicited by various environmental and endogenous factors. They bear the risk of cell lethality and, if mis-repaired, of deleterious mutation. This negative impact is contrasted by several evolutionary achievements for DSB processing that help maintaining stable inheritance (correct repair, meiotic cross-over) and even drive adaptation (immunoglobulin gene recombination), differentiation (chromatin elimination) and speciation by creating new genetic diversity via DSB mis-repair. Targeted DSBs play a role in genome editing for research, breeding and therapy purposes. Here, I survey possible causes, biological effects and evolutionary consequences of DSBs, mainly for students and outsiders.

Stochastic multicellular modeling of x-ray irradiation, DNA damage induction, DNA free-end misrejoining and cell death

The repair or misrepair of DNA double-strand breaks (DSBs) largely determines whether a cell will survive radiation insult or die. A new computational model of multicellular, track structure-based and pO2-dependent radiation-induced cell death was developed and used to investigate the contribution to cell killing by the mechanism of DNA free-end misrejoining for low-LET radiation. A simulated tumor of 1224 squamous cells was irradiated with 6 MV x-rays using the Monte Carlo toolkit Geant4 with low-energy Geant4-DNA physics and chemistry modules up to a uniform dose of 1 Gy. DNA damage including DSBs were simulated from ionizations, excitations and hydroxyl radical interactions along track segments through cell nuclei, with a higher cellular pO2 enhancing the conversion of DNA radicals to strand breaks. DNA free-ends produced by complex DSBs (cDSBs) were able to misrejoin and produce exchange-type chromosome aberrations, some of which were asymmetric and lethal. A sensitivity analysis was performed and conditions of full oxia and anoxia were simulated. The linear component of cell killing from misrejoining was consistently small compared to values in the literature for the linear component of cell killing for head and neck squamous cell carcinoma (HNSCC). This indicated that misrejoinings involving DSBs from the same x-ray (including all associated secondary electrons) were rare and that other mechanisms (e.g. unrejoined ends) may be important. Ignoring the contribution by the indirect effect toward DNA damage caused the DSB yield to drop to a third of its original value and the cDSB yield to drop to a tenth of its original value. Track structure-based cell killing was simulated in all 135306 viable cells of a 1 mm3 hypoxic HNSCC tumor for a uniform dose of 1 Gy.

Genetic interaction between DNA repair factors PAXX, XLF, XRCC4 and DNA-PKcs in human cells.

DNA double‐strand breaks (DSBs) are highly cytotoxic lesions, and unrepaired or misrepaired DSBs can lead to various human diseases, including immunodeficiency, neurological abnormalities, growth retardation, and cancer. Nonhomologous end joining (NHEJ) is the major DSB repair pathway in mammals. Ku70 and Ku80 are DSB sensors that facilitate the recruitment of downstream factors, including protein kinase DNA‐dependent protein kinase, catalytic subunit (DNA‐PKcs), structural components [X‐ray repair cross‐complementing protein 4 (XRCC4), XRCC4‐like factor (XLF), and paralogue of XRCC4 and XLF (PAXX)], and DNA ligase IV (LIG4), which complete DNA repair. DSBs also trigger the activation of the DNA damage response pathway, in which protein kinase ataxia‐telangiectasia mutated (ATM) phosphorylates multiple substrates, including histone H2AX. Traditionally, research on NHEJ factors was performed using in vivo mouse models and murine cells. However, the current knowledge of the genetic interactions between NHEJ factors in human cells is incomplete. Here, we obtained genetically modified human HAP1 cell lines, which lacked one or two NHEJ factors, including LIG4, XRCC4, XLF, PAXX, DNA‐PKcs, DNA‐PKcs/XRCC4, and DNA‐PKcs/PAXX. We examined the genomic instability of HAP1 cells, as well as their sensitivity to DSB‐inducing agents. In addition, we determined the genetic interaction between XRCC4 paralogues (XRCC4, XLF, and PAXX) and DNA‐PKcs. We found that in human cells, XLF, but not PAXX or XRCC4, genetically interacts with DNA‐PKcs. Moreover, ATM possesses overlapping functions with DNA‐PKcs, XLF, and XRCC4, but not with PAXX in response to DSBs. Finally, NHEJ‐deficient HAP1 cells show increased chromosomal and chromatid breaks, when compared to the WT parental control. Overall, we found that HAP1 is a suitable model to study the genetic interactions in human cells.

Genome-Wide Analysis of Interchromosomal Interaction Probabilities Reveals Chained Translocations and Overrepresentation of Translocation Breakpoints in Genes in a Cutaneous T-Cell Lymphoma Cell Line.

In classical models of tumorigenesis, the accumulation of tumor promoting chromosomal aberrations is described as a gradual process. Next-generation sequencing-based methods have recently revealed complex patterns of chromosomal aberrations, which are beyond explanation by these classical models of karyotypic evolution of tumor genomes. Thus, the term chromothripsis has been introduced to describe a phenomenon, where temporarily and spatially confined genomic instability results in dramatic chromosomal rearrangements limited to segments of one or a few chromosomes. Simultaneously arising and misrepaired DNA double-strand breaks are also the cause of another phenomenon called chromoplexy, which is characterized by the presence of chained translocations and interlinking deletion bridges involving several chromosomes. In this study, we demonstrate the genome-wide identification of chromosomal translocations based on the analysis of translocation-associated changes in spatial proximities of chromosome territories on the example of the cutaneous T-cell lymphoma cell line Se-Ax. We have used alterations of intra- and interchromosomal interaction probabilities as detected by genome-wide chromosome conformation capture (Hi-C) to infer the presence of translocations and to fine-map their breakpoints. The outcome of this analysis was subsequently compared to datasets on DNA copy number alterations and gene expression. The presence of chained translocations within the Se-Ax genome, partly connected by intervening deletion bridges, indicates a role of chromoplexy in the etiology of this cutaneous T-cell lymphoma. Notably, translocation breakpoints were significantly overrepresented in genes, which highlight gene-associated biological processes like transcription or other gene characteristics as a possible cause of the observed complex rearrangements. Given the relevance of chromosomal aberrations for basic and translational research, genome-wide high-resolution analysis of structural chromosomal aberrations will gain increasing importance.

More end resection is not merrier.

Unrestrained 53BP1 activity causes fusions of dysfunctional telomeres and embryonic lethality associated with misrepair of DNA double-strand breaks in BRCA1-deficient mice. However, the physiological role of 53BP1 remains unclear, because it presumably did not evolve to carry out these pathological functions. A new report proposes that 53BP1 activity prevents hyper-resection and thereby promotes error-free DNA repair while suppressing alternative mutagenic pathways.

Expression of MLL-AF4 or AF4-MLL fusions does not impact the efficiency of DNA damage repair.

The most frequent rearrangement of the human MLL gene fuses MLL to AF4 resulting in high-risk infant B-cell acute lymphoblastic leukemia (B-ALL). MLL fusions are also hallmark oncogenic events in secondary acute myeloid leukemia. They are a direct consequence of mis-repaired DNA double strand breaks (DNA-DSBs) due to defects in the DNA damage response associated with exposure to topoisomerase-II poisons such as etoposide. It has been suggested that MLL fusions render cells susceptible to additional chromosomal damage upon exposure to etoposide. Conversely, the genome-wide mutational landscape in MLL-rearranged infant B-ALL has been reported silent. Thus, whether MLL fusions compromise the recognition and/or repair of DNA damage remains unanswered. Here, the fusion proteins MLL-AF4 (MA4) and AF4-MLL (A4M) were CRISPR/Cas9-genome edited in the AAVS1 locus of HEK293 cells as a model to study MLL fusion-mediated DNA-DSB formation/repair. Repair kinetics of etoposide- and ionizing radiation-induced DSBs was identical in WT, MA4- and A4M-expressing cells, as revealed by flow cytometry, by immunoblot for γH2AX and by comet assay. Accordingly, no differences were observed between WT, MA4- and A4M-expressing cells in the presence of master proteins involved in non-homologous end-joining (NHEJ; i.e.KU86, KU70), alternative-NHEJ (Alt-NHEJ; i.e.LigIIIa, WRN and PARP1), and homologous recombination (HR, i.e.RAD51). Moreover, functional assays revealed identical NHEJ and HR efficiency irrespective of the genotype. Treatment with etoposide consistently induced cell cycle arrest in S/G2/M independent of MA4/A4M expression, revealing a proper activation of the DNA damage checkpoints. Collectively, expression of MA4 or A4M does neither influence DNA signaling nor DNA-DSB repair.

Repair Pathway Choices and Consequences at the Double-Strand Break.

DNA double-strand breaks (DSBs) are cytotoxic lesions that threaten genomic integrity. Failure to repair a DSB has deleterious consequences, including genomic instability and cell death. Indeed, misrepair of DSBs can lead to inappropriate end-joining events, which commonly underlie oncogenic transformation due to chromosomal translocations. Typically, cells employ two main mechanisms to repair DSBs: homologous recombination (HR) and classical nonhomologous end joining (C-NHEJ). In addition, alternative error-prone DSB repair pathways, namely alternative end joining (alt-EJ) and single-strand annealing (SSA), have been recently shown to operate in many different conditions and to contribute to genome rearrangements and oncogenic transformation. Here, we review the mechanisms regulating DSB repair pathway choice, together with the potential interconnections between HR and the annealing-dependent error-prone DSB repair pathways.

Cell transcriptional state alters genomic patterns of DNA double-strand break repair in human astrocytes.

The misrepair of DNA double-strand breaks in close spatial proximity within the nucleus can result in chromosomal rearrangements that are important in the pathogenesis of haematopoietic and solid malignancies. It is unknown why certain epigenetic states, such as those found in stem or progenitor cells, appear to facilitate neoplastic transformation. Here we show that altering the transcriptional state of human astrocytes alters patterns of DNA damage repair from ionizing radiation at a gene locus-specific and genome-wide level. Astrocytes induced into a reactive state exhibit increased DNA repair, compared with non-reactive cells, in actively transcribed chromatin after irradiation. In mapping these repair sites, we identify misrepair events and repair hotspots that are unique to each state. The precise characterization of genomic regions susceptible to mutation in specific transcriptional states provides new opportunities for addressing clonal evolution in solid cancers, in particular those where double-strand break induction is a cornerstone of clinical intervention.

Multicolour FISH analysis of ionising radiation induced micronucleus formation in human lymphocytes.

Micronucleation of chromosomal DNA is an effective indicator of DNA damage and micronucleus (MN) analysis is a valuable tool for radiation biodosimetry studies. To gain a comprehensive knowledge of micronucleation process after ionising radiation (IR) exposure, whole genome-wide chromosome analysis is desirable. With this objective, multicolour fluorescence in situ hybridization (M-FISH) technique was utilised in the present study to characterise the chromosome content of spontaneous and IR-induced micronuclei in three human donors. M-FISH analysis revealed a radiation dose-dependant increase in the number of micronuclei with multi-chromosome material above 2 Gy and as many as 3-6 multicolour signals were detected in micronuclei after high γ-rays radiation doses (5-10 Gy). Involvement of each human chromosome material was more frequently detected in multicoloured micronuclei than in single-coloured micronuclei at high radiation doses (>2 Gy). Observation of dose-dependant increase in the MN frequency with multi-chromosome material may be due to misrepair of DNA double-strand breaks involving multiple chromosomes leading to asymmetric dicentric or ring chromosomes and acentric fragments. Chromosomes belonging to groups A (1, 2 and 3) and B (4 and 5) were frequently detected in 35-45% of the total micronuclei either as single entities or in combination with other chromosomes. Among the A and B groups, chromosome 1 material was consistently detected at high MN frequencies after radiation exposure in all the donors. Additionally, chromosomes 13 and 19 were more frequently observed in micronuclei than the expected frequency based on DNA content. Our whole genome approach utilising the M-FISH technique revealed that MN formation at high radiation doses might be complex involving multiple chromosome fragments. Understanding the fate and biological consequences of these multi-chromosome-containing micronuclei may provide key molecular insights for some aspects of IR-induced genomic instability and cancer development processes.

Targeting components of the alternative NHEJ pathway sensitizes KRAS mutant leukemic cells to chemotherapy

AbstractActivating KRAS mutations are detected in a substantial number of hematologic malignancies. In a murine T-cell acute lymphoblastic leukemia (T-ALL) model, we previously showed that expression of oncogenic Kras induced a premalignant state accompanied with an arrest in T-cell differentiation and acquisition of somatic Notch1 mutations. These findings prompted us to investigate whether the expression of oncogenic KRAS directly affects DNA damage repair. Applying divergent, but complementary, genetic approaches, we demonstrate that the expression of KRAS mutants is associated with increased expression of DNA ligase 3α, poly(ADP-ribose) polymerase 1 (PARP1), and X-ray repair cross-complementing protein 1 (XRCC1), all essential components of the error-prone, alternative nonhomologous end-joining (alt-NHEJ) pathway. Functional studies revealed delayed repair kinetics, increased misrepair of DNA double-strand breaks, and the preferential use of microhomologous DNA sequences for end joining. Similar effects were observed in primary murine T-ALL blasts. We further show that KRAS-mutated cells, but not KRAS wild-type cells, rely on the alt-NHEJ repair pathway on genotoxic stress. RNA interference–mediated knockdown of DNA ligase 3α abolished resistance to apoptotic cell death in KRAS-mutated cells. Our data indicate that targeting components of the alt-NHEJ pathway sensitizes KRAS-mutated leukemic cells to standard chemotherapeutics and represents a promising approach for inducing synthetic lethal vulnerability in cells harboring otherwise nondruggable KRAS mutations.

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.

Misrepair of DNA double-strand breaks after exposure to heavy-ion beams causes a peak in the LET–RBE relationship with respect to cell killing in DT40 cells

To determine the radiobiological mechanisms underlying relative biological effectiveness (RBE) and the repair efficiencies of DNA double-strand breaks (DSBs) as a function of linear energy transfer (LET), we exposed cells of the chicken B-lymphocyte cell line DT40 and its DSB repair pathway-deficient derivatives to heavy-ion beams produced at the Heavy-Ion Medical Accelerator in Chiba (HIMAC) at the National Institute of Radiological Sciences (NIRS), Chiba, Japan. The relationship between LET and cell lethality was investigated in the DNA DSB repair gene knockouts Ku70−/−, Rad54−/−, and Ku70−/−Rad54−/−, and in the wild-type cells. We found that cell-cycle stage and activity of the DNA DSB repair pathways influence LET-mediated biological effects. An expected LET–RBE relationship was observed in the cells capable of DNA repair, but no peak was found in the RBE with respect to cell survival in the Ku70−/−Rad54−/− cells or in Ku70−/− cells in the G1 and early S cell-cycle phases (when no sister chromatids were present and homologous recombination could not occur). These findings suggest that the peak in RBE is caused by deficient repair of the DNA DSBs.

Accumulation of DNA Damage-Induced Chromatin Alterations in Tissue-Specific Stem Cells: The Driving Force of Aging?

Accumulation of DNA damage leading to stem cell exhaustion has been proposed to be a principal mechanism of aging. Using 53BP1-foci as a marker for DNA double-strand breaks (DSBs), hair follicle stem cells (HFSCs) in mouse epidermis were analyzed for age-related DNA damage response (DDR). We observed increasing amounts of 53BP1-foci during the natural aging process independent of telomere shortening and after protracted low-dose radiation, suggesting substantial accumulation of DSBs in HFSCs. Electron microscopy combined with immunogold-labeling showed multiple small 53BP1 clusters diffusely distributed throughout the highly compacted heterochromatin of aged HFSCs, but single large 53BP1 clusters in irradiated HFSCs. These remaining 53BP1 clusters did not colocalize with core components of non-homologous end-joining, but with heterochromatic histone modifications. Based on these results we hypothesize that these lesions were not persistently unrepaired DSBs, but may reflect chromatin rearrangements caused by the repair or misrepair of DSBs. Flow cytometry showed increased activation of repair proteins and damage-induced chromatin modifications, triggering apoptosis and cellular senescence in irradiated, but not in aged HFSCs. These results suggest that accumulation of DNA damage-induced chromatin alterations, whose structural dimensions reflect the complexity of the initial genotoxic insult, may lead to different DDR events, ultimately determining the biological outcome of HFSCs. Collectively, our findings support the hypothesis that aging might be largely the remit of structural changes to chromatin potentially leading to epigenetically induced transcriptional deregulation.

Chromosomal instability induced by mammography X-rays in primary human fibroblasts from BRCA1 and BRCA2 mutation carriers

Purpose: Mammography X-rays are known to induce DNA double-strand breaks (DSB) whose error-free recombinational repair requires the function of the tumour repressor genes BRCA1 (breast-cancer-associated gene 1) and BRCA2 (breast-cancer-associated gene 2). Since un- or misrepaired DSB lead to chromosomal anomalies which may promote the development of breast cancer, we have studied the potential of mammography X-rays for immediate and delayed induction of chromosomal anomalies in human primary fibroblasts from BRCA1 and BRCA2 mutation carriers.Materials and methods: Primary human fibroblasts from three BRCA1, three BRCA2 mutation carriers, one BRCA2-deficient fanconi anemia (FA) patient and three normal individuals were exposed to various doses of mammography X-rays. Chromosomal anomalies at first mitosis and at several population doublings post-irradiation were assayed (Giemsa staining and Fish [fluorescence in situ hybridization]).Results: No effect of the BRCA mutation status was observed on survival curves after exposure to mammography X-rays and on the dose-dependent increase of chromosomal anomalies at first mitosis post-irradiation. In contrast, several population doublings after exposure to a low dose of only 0.5 Gy chromosomal instability, manifested as gross chromosomal rearrangements and aneuploidy, had developed in BRCA2-deficient FA fibroblasts and in some – but not all – BRCA heterozygous fibroblasts.Conclusions: Low doses of mammography X-rays have the potential to induce chromosomal instability in fibroblasts from BRCA mutation carriers: Cells exhibit gross chromosomal rearrangements and aneuploidy similar to those observed in breast cancer cells. These results suggest that for women carrying a BRCA mutation early and frequent screening with mammography X-rays may not be the method of choice to detect breast cancer.

Accumulation of DNA damage in complex normal tissues after protracted low-dose radiation

The biological consequences of low levels of radiation exposure and their effects on human health are unclear. Ionizing radiation induces a variety of lesions of which DNA double-strand breaks (DSBs) are the most biologically significant, because unrepaired or misrepaired DSBs can lead to genomic instability and cell death. Using repair-proficient mice as an in vivo system we monitored the accumulation of DNA damage in normal tissues exposed to daily low-dose radiation of 100mGy or 10mGy. Radiation-induced foci in differentiated and tissue-specific stem cells were quantified by immunofluorescence microscopy after 2, 4, 6, 8, and 10 weeks of daily low-dose radiation and DNA lesions were characterized using transmission electron microscopy (TEM) combined with immunogold-labeling. In brain, long-living cortical neurons had a significant accumulation of foci with increasing cumulative doses. In intestine and skin, characterized by constant cell renewal of their epithelial lining, differentiated enterocytes and keratinocytes had either unchanged or only slightly increased foci levels during protracted low-dose radiation. Significantly, analysis of epidermal stem cells in skin revealed a constant increase of 53BP1 foci during the first weeks of low-dose radiation even with 10mGy, suggesting substantial accumulations of DSBs. However, TEM analysis suggests that these remaining 53BP1 foci, which are predominantly located in compact heterochromatin, do not co-localize with phosphorylated Ku70 or DNA-PKcs, core components of non-homologous end-joining. The biological relevance of these persistent 53BP1 foci, particularly their contribution to genomic instability by genetic and epigenetic alterations, has to be defined in future studies.

Genotoxicity of uranium contamination in embryonic zebrafish cells

Uranium is a metal used in the nuclear industry and for military applications. Studies on mammals have shown that uranium is genotoxic. However the molecular and cellular mechanisms responsible for the genotoxicity of uranium are poorly known for other types of vertebrates such as fish. Since unrepaired DNA double-strand breaks (DSBs) are considered to be key lesions in cell lethality, the activity of one of the major DSB-repair pathways, i.e. non-homologous end-joining (NHEJ), has been evaluated in embryonic zebrafish cells (ZF4) exposed to uranium. Genotoxicity of uranium in ZF4 cells was further assessed by comet and micronucleus assays. Exposure to uranium results in the production of DSBs a few hours after incubation. These breaks trigger the phosphorylation of H2AX proteins. We showed that the DNA-PK kinase activity, essential for NHEJ, is altered by the presence of uranium. The presence of uranium in cells disturbs but does not inhibit the repair rate of DSBs. Such a result suggests an impact of uranium upon the reparability of DSBs and the potential activation of alternative DSBs repair pathway leading to the propagation of possible misrepaired DSBs. In parallel, we performed a transmission electron microscopy analysis of cells exposed to uranium and were able to localize internalized uranium using an Energy Dispersive X-ray microanalyser. We observed the formation of precipitates in lysosome-like vesicles for 250μM of uranium in the medium. The appearance of these precipitates is concomitant with the decrease of the number of DSBs per cell. This process might be a part of a defence system whose role in counteracting cytotoxicity calls for further dedicated research.

The RAG2 C terminus suppresses genomic instability and lymphomagenesis

Misrepair of DNA double-strand breaks (DSBs) produced by the V(D)J recombinase (the RAG1/RAG2 proteins) at immunoglobulin (Ig) and T cell receptor (Tcr) loci has been implicated in pathogenesis of lymphoid malignancies in humans1 and in mice2–7. Defects in DNA damage response factors such as ATM and combined deficiencies in classical nonhomologous end joining (NHEJ) and p53 predispose to RAG-initiated genomic rearrangements and lymphomagenesis2–11. Although we showed previously that RAG1/RAG2 shepherd the broken DNA ends to classical NHEJ for proper repair12,13, roles for the RAG proteins in preserving genomic stability remain poorly defined. Here we show that the RAG2 C-terminus, although dispensable for recombination14,15, is critical for maintaining genomic stability. Thymocytes from “core” Rag2 homozygotes (Rag2c/c mice) show dramatic disruption of Tcrα/δ locus integrity. Furthermore, all Rag2c/c p53−/− mice, unlike Rag1c/c p53−/− and p53−/− animals, rapidly develop thymic lymphomas bearing complex chromosomal translocations, amplifications, and deletions involving the Tcrα/δ and Igh loci. We also find these features in lymphomas from Atm−/− mice. We show that, like ATM-deficiency3, core RAG2 severely destabilizes the RAG post-cleavage complex. These results reveal a novel genome guardian role for RAG2 and suggest that similar “end release/end persistence” mechanisms underlie genomic instability and lymphomagenesis in Rag2c/c p53−/− and Atm−/− mice.

MRE11 and H2AX biomarkers in the response to low-dose exposure: balance between individual susceptibility to radiosensitivity and to genomic instability

Unrepaired and misrepaired DNA Double-Strand Breaks (DSBs) were shown to be the key events of radiation-induced toxicity (radiosensitivity) and genomic instability (cancer proneness), respectively. We have previously shown that immunofluorescence with γ -H2AX and MRE11 biomarkers may account for unrepaired and misrepaired DSBs for doses higher than 1 Gy. Three radiosensitivity groups had been defined (group I: radioresistance; group II: moderate radiosensitivity and cancer proneness; group III: hyper-radiosensitivity). Here, we investigated X-ray doses ranging from 9 mGy to 2 Gy in three cell lines representative of these radiosensitivity groups. We observed a non-linear dose-dependent increase of the severity of DSB, suggesting a correlation with the phenomenon of hyper-radiosensitivity to low dose; a non-linear dose-dependent increase of MRE11 foci, revealing a dose-threshold for the radiation-induced genomic instability. Altogether, these data document the evidence of a threshold in low-dose response and strongly suggest the impact of individual factor.