Purpose: Higher order chromatin structure progressively changes with cell differentiation and seems to play an important role in DNA double-strand break (DSB) induction and repair (reviewed in ). We compared DNA damage in heterochromatin (Hc) upon the action of qualitatively different radiations. We also studied, how is the sensitivity to DSB induction, assembly of repair foci and processing of DSBs influenced by the differentiation-induced changes in chromatin structure and composition.
Materials and methods: Formation, localization (relative to higher-order chromatin domains) and mutual colocalization of γH2AX and p53BP1 repair foci have been studied together with DSB repair kinetics in spatially fixed human skin fibroblast and differently differentiated white blood cells (WBC) irradiated with gamma rays, protons of different energies [2, 3], and 20Ne ions (submitted). Immunostaining and ImmunoFISH were used in combination with high-resolution confocal microscopy [2, 3] and living cell imaging .
Results: We found that less DSBs appear in Hc after irradiating cells with gamma rays and protons but not 20Ne ions (preliminary results). In addition, contrary to γ-irradiated human skin fibroblasts and lymphocytes, mature granulocytes neither express DSB repair proteins nor form functional repair foci . At least some DSB repair proteins (e.g. 53BP1) are expressed and γH2AX foci still occur in immature granulocytes and monocytes [2, 5]; however, the colocalization of γH2AX with 53BP1 is low and the majority of DSBs are not repaired. Despite this fact, γH2AX foci protrude from Hc into nuclear subcompartments with low chromatin density. Our living cell observations suggest that 53BP1 can penetrate into the interior of dense Hc domains only after their decondensation .
Conclusions: We show that Hc is less sensitive to DSB induction by gamma rays but not heavy ions; lower Hc hydratation and higher protein density (when compared with euchromatin) probably reduce formation of free radicals and increase their sequestration, respectively. This mechanism can protect cells against the indirect effect of ionizing radiation (marked for gamma rays and protons but not heavy ions). Hc features, however, preclude DSB repair, which is best illustrated by its absence in differentiated WBC but not their immature precursors. The protrusion of Hc-DSBs into low-density chromatin nuclear subdomains, however, appears also in differentiated WBC, so the process might simply follow physical forces (e.g. as suggested by M Durante's group).
There is no Clinical Trial Registration number.
DNA double-strand breaks (DSB); DSB repair; white blood cells differentiation; higher-order chromatin structure; ionizing radiations of different quality; ionizing radiation-induced repair foci (IRIF)
The signature DNA lesion induced by ionizing radiation is clustered DNA damage. Gamma radiation-induced clustered DNA damage containing base lesions was investigated in plasmid DNA under cell mimetic conditions and in two cell lines, V79-4 (hamster) and HF19 (human), using bacterial endonucleases Nth (endonuclease III) and Fpg (formamidopyrimidine DNA glycosylase). Following irradiation with 60Co γ-rays, induction of double-strand breaks (DSB) and clustered DNA damage, revealed as DSB by the proteins, was determined in plasmid using the plasmid-nicking assay and in cells by either conventional pulsed field gel electrophoresis or a hybridization assay, in which a 3 Mb restriction fragment of the X chromosome is used as a radioactive labeled probe. Enzyme concentrations (30–60 ng/µg DNA) were optimized to minimize visualization of background levels of endogenous DNA damage and DSB produced by non-specific cutting by Fpg and Nth in cellular DNA. 60Co γ- radiation produces a 1.8-fold increase in the yields of both types of enzyme sensitive sites, visualized as DSB compared with that of prompt DSB in plasmid DNA. In mammalian cells, the increase in yields of clustered DNA damage containing either Fpg or Nth sensitive sites compared with that of prompt DSB is 1.4–2.0- and 1.8-fold, respectively. Therefore, clustered DNA damage is induced in cells by sparsely ionizing radiation and their yield is significantly greater than that of prompt DSB.
Double-strand break (DSB) repair through homologous recombination (HR) is an evolutionarily conserved process that is generally error-free. The risk to genome stability posed by nonallelic recombination or loss-of-heterozygosity could be reduced by confining HR to sister chromatids, thereby preventing recombination between homologous chromosomes. Here we show that the sister chromatid cohesion complex (cohesin) is a limiting factor in the control of DSB repair and genome stability and that it suppresses DNA damage–induced interactions between homologues. We developed a gene dosage system in tetraploid yeast to address limitations on various essential components in DSB repair and HR. Unlike RAD50 and RAD51, which play a direct role in HR, a 4-fold reduction in the number of essential MCD1 sister chromatid cohesion subunit genes affected survival of gamma-irradiated G2/M cells. The decreased survival reflected a reduction in DSB repair. Importantly, HR between homologous chromosomes was strongly increased by ionizing radiation in G2/M cells with a single copy of MCD1 or SMC3 even at radiation doses where survival was high and DSB repair was efficient. The increased recombination also extended to nonlethal doses of UV, which did not induce DSBs. The DNA damage–induced recombinants in G2/M cells included crossovers. Thus, the cohesin complex has a dual role in protecting chromosome integrity: it promotes DSB repair and recombination between sister chromatids, and it suppresses damage-induced recombination between homologues. The effects of limited amounts of Mcd1and Smc3 indicate that small changes in cohesin levels may increase the risk of genome instability, which may lead to genetic diseases and cancer.
The cellular concentrations of individual proteins are expected to be kept within an optimal range, but protein expression is often stochastic. Some proteins are known to be in limiting amounts, so that even modest reduction can lead to malfunction. Within the network of genes that determine genome stability, proteins that are limiting impose a risk for the cell, because fluctuation in their amounts may start a cascade of genomic alternations that will influence many biochemical pathways either under normal growth conditions or in response to chromosome damage. We sought to identify genes that are limiting for DSB repair by lowering the dosage of key genes from 4 to 1 in tetraploid Saccharomyces cerevisiae strains. We found that the complex that holds sister chromatid cohesion together (cohesin) is limiting in DSB repair. In addition, when it is reduced modestly, recombination between homologous chromosomes is highly increased, suggesting that the risk for loss of hetrozygosity (LOH) is increased too. These results should also be considered in light of increasing evidence that copy number variation can impact cellular function.
We describe a new assay for in vitro repair
of oxidatively induced DNA double-strand breaks (DSBs) by HeLa cell
nuclear extracts. The assay employs linear plasmid DNA containing
DNA DSBs produced by the radiomimetic drug bleomycin. The bleomycin-induced
DSB possesses a complex structure similar to that produced by oxidative
processes and ionizing radiation. Bleomycin DSBs are composed of
blunt ends or ends containing a single 5′-base
overhang. Regardless of the 5′-end structure,
all bleomycin-induced DSBs possess 3′-ends
blocked by phosphoglycolate. Cellular extraction and initial end joining
conditions for our assay were optimized with restriction enzyme-cleaved
DNA to maximize ligation activity. Parameters affecting ligation
such as temperature, time, ionic strength, ATP utilization and extract
protein concentration were examined. Similar reactions were performed
with the bleomycin-linearized substrate. In all cases, end-joined molecules
ranging from dimers to higher molecular weight forms were produced
and observed directly in agarose gels stained with Vistra Green
and imaged with a FluorImager 595. This detection method is at least
50-fold more sensitive than ethidium bromide and permits detection
of ≤0.25 ng double-stranded DNA per
band in post-electrophoretically stained agarose gels. Consequently,
our end-joining reaction requires ≤100
ng substrate DNA and ≥50% conversion
of substrate to product is achieved with simple substrates such
as restriction enzyme-cleaved DNA. Using our assay we have observed
a 6-fold lower repair rate and a lag in reaction initiation for bleomycin-induced
DSBs as compared to blunt-ended DNA. Also, end joining reaction
conditions are DSB end group dependent. In particular, bleomycin-induced
DSB repair is considerably more sensitive to inhibition by increased
ionic strength than repair of blunt-ended DNA.
DNA double-strand breaks (DSBs) are potent sources of genome instability. While there is considerable genetic and molecular information about the disposition of direct DSBs and breaks that arise during replication, relatively little is known about DSBs derived during processing of single-strand lesions, especially for the case of single-strand breaks (SSBs) with 3′-blocked termini generated in vivo. Using our recently developed assay for detecting end-processing at random DSBs in budding yeast, we show that single-strand lesions produced by the alkylating agent methyl methanesulfonate (MMS) can generate DSBs in G2-arrested cells, i.e., S-phase independent. These derived DSBs were observed in apn1/2 endonuclease mutants and resulted from aborted base excision repair leading to 3′ blocked single-strand breaks following the creation of abasic (AP) sites. DSB formation was reduced by additional mutations that affect processing of AP sites including ntg1, ntg2, and, unexpectedly, ogg1, or by a lack of AP sites due to deletion of the MAG1 glycosylase gene. Similar to direct DSBs, the derived DSBs were subject to MRX (Mre11, Rad50, Xrs2)-determined resection and relied upon the recombinational repair genes RAD51, RAD52, as well as on the MCD1 cohesin gene, for repair. In addition, we identified a novel DNA intermediate, detected as slow-moving chromosomal DNA (SMD) in pulsed field electrophoresis gels shortly after MMS exposure in apn1/2 cells. The SMD requires nicked AP sites, but is independent of resection/recombination processes, suggesting that it is a novel structure generated during processing of 3′-blocked SSBs. Collectively, this study provides new insights into the potential consequences of alkylation base damage in vivo, including creation of novel structures as well as generation and repair of DSBs in nonreplicating cells.
DNA double-strand breaks (DSBs) are an important source of genome instability that can lead to severe biological consequences including tumorigenesis and cell death. Although much is known about DSBs induced directly by ionizing radiation and radiomimetic cancer drugs, there is a relative dearth of information about the formation of derived DSBs that arise from processing of single-strand lesions. Since as many as 10,000–200,000 single-strand lesions have been estimated to occur each day in mammalian cells, conversion of even a small percentage of such lesions to DSBs could dramatically affect genome stability. Here we addressed the mechanism of formation and repair of derived DSBs in vivo during the processing of DNA methylation damage in yeast that are defective in base excision repair (BER) due to a lack of AP endonucleases. Armed with a technique developed in our lab that detects resection at DSBs, a first step in DSB repair, we demonstrated formation of DSBs in G2 cells and the role of recombinational repair in subsequent chromosome restitution. Furthermore, we have identified a novel repair intermediate that can be generated if abasic sites are nicked by AP lyases, providing additional insights into the processing of 3′-blocked groups at single-strand breaks.
The protein kinase CK2 sustains multiple pro-survival functions in cellular DNA damage response and its level is tightly regulated in normal cells but elevated in cancers. Because CK2 is thus considered as potential therapeutic target, DNA double-strand break (DSB) formation and rejoining, apoptosis induction and clonogenic survival was assessed in irradiated mammalian cells upon chemical inhibition of CK2.
MRC5 human fibroblasts and WIDR human colon carcinoma cells were incubated with highly specific CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB), or mock-treated, 2 hours prior to irradiation. DSB was measured by pulsed-field electrophoresis (PFGE) as well as gamma-H2AX foci formation and removal. Apoptosis induction was tested by DAPI staining and sub-G1 flow cytometry, survival was quantified by clonogenic assay.
TBB treatment did not affect initial DNA fragmention (PFGE; up to 80 Gy) or foci formation (1 Gy). While DNA fragment rejoining (PFGE) was not inhibited by the drug, TBB clearly delayed gamma-H2AX foci disappearence during postirradiation incubation. No apoptosis induction could be detected for up to 38 hours for both cell lines and exposure conditions (monotherapies or combination), but TBB treatment at this moderately toxic concentration of 20 μM (about 40% survival) enhanced radiation-induced cell killing in the clonogenic assay.
The data imply a role of CK2 in gamma-H2AX dephosporylation, most likely through its known ability to stimulate PP2A phosphatase, rather than DSB rejoining. The slight but definite clonogenic radiosensitization by TBB does apparently not result from interference with an apoptosis suppression function of CK2 in these cells but could reflect inhibitor-induced uncoupling of DNA damage response decay from break ligation.
Head and neck cancers (head and neck squamous cell carcinomas [HNSCC]) are a heterogeneous group of neoplasms with varying presenting symptoms, treatment, and expected outcome. There is a need to find an effective way of its treatment at the molecular level. Thus, we should identify the mechanism of cancer cell response to damaging agents' activity, especially at DNA level. Our major goal was to evaluate the efficacy of DNA double strand breaks (DSBs) repair in HTB-43 and SCC-25 cancer cell lines as well as lymphocytes taken from HNSCC patients and healthy donors. The DNA repair efficiency was measured by neutral comet assay as well as extrachromosomal assay for DNA DSBs repair (TAK assay). We determined the levels of two main pathways of DNA DSBs—nonhomologous end joining (NHEJ) and homologous recombination repair (HRR). Neutral comet assay was used for evaluation of DNA DSBs repair after treatment with genotoxic agents. DNA DSBs induced by gamma radiation were repaired slower in lymphocytes from HNSCC patients than in lymphocytes from healthy controls. HTB-43 and SCC-25 cancer cell lines have higher efficacy of NHEJ and HRR than lymphocytes taken from patients as well as control subjects. Our results confirm the necessity of further studies on the mechanisms of DNA DSBs repair to provide insight into the molecular basis of head and neck cancer, which will allow us to improve methods of HNSCC treatment.
Resection of DNA double-strand break (DSB) ends is generally considered a critical determinant in pathways of DSB repair and genome stability. Unlike for enzymatically induced site-specific DSBs, little is known about processing of random “dirty-ended” DSBs created by DNA damaging agents such as ionizing radiation. Here we present a novel system for monitoring early events in the repair of random DSBs, based on our finding that single-strand tails generated by resection at the ends of large molecules in budding yeast decreases mobility during pulsed field gel electrophoresis (PFGE). We utilized this “PFGE-shift” to follow the fate of both ends of linear molecules generated by a single random DSB in circular chromosomes. Within 10 min after γ-irradiation of G2/M arrested WT cells, there is a near-synchronous PFGE-shift of the linearized circular molecules, corresponding to resection of a few hundred bases. Resection at the radiation-induced DSBs continues so that by the time of significant repair of DSBs at 1 hr there is about 1–2 kb resection per DSB end. The PFGE-shift is comparable in WT and recombination-defective rad52 and rad51 strains but somewhat delayed in exo1 mutants. However, in rad50 and mre11 null mutants the initiation and generation of resected ends at radiation-induced DSB ends is greatly reduced in G2/M. Thus, the Rad50/Mre11/Xrs2 complex is responsible for rapid processing of most damaged ends into substrates that subsequently undergo recombinational repair. A similar requirement was found for RAD50 in asynchronously growing cells. Among the few molecules exhibiting shift in the rad50 mutant, the residual resection is consistent with resection at only one of the DSB ends. Surprisingly, within 1 hr after irradiation, double-length linear molecules are detected in the WT and rad50, but not in rad52, strains that are likely due to crossovers that are largely resection- and RAD50-independent.
Double-strand breaks (DSBs) in chromosomal DNA are common sources of genomic change that may be beneficial or deleterious to an organism, from yeast to humans. While they can arise through programmed cellular events, DSBs are frequently associated with defective chromosomal replication, and they are induced by various types of DNA damaging agents such as those employed in cancer therapy, especially ionizing radiation. Elaborate systems have evolved for DSB recognition and subsequent repair, either by homologous recombination or by direct joining of ends. Although much is known about repair mechanisms associated with defined, artificially produced DSBs, there is a relative dearth of information about events surrounding random DSBs. Using a novel, yeast-based system that is applicable to other organisms, we have addressed resection at DSBs, considered a first step in repair. We provide the first direct evidence that cells possess a highly efficient system for recognition and initiation of resection at γ-radiation–induced dirty ends and that the resection is largely dependent on the Rad50/Mre11/Xrs2 complex, identified by the RAD50 gene. The system provides unique opportunities to address other components in resection and repair as well as to identify the contribution of random DSBs and resection to genome instability resulting from other DNA damaging agents.
Heavy ion beam as typical high linear energy transfer (LET) radiation produces more expanding ionization domain around their tracks than low LET radiation such as X-rays and gamma rays. Thus, heavy ion beam can cause more densely accumulated damage cluster in the target DNA, termed clustered DNA damage. This damage exhibits difficulty for repair and inhibition of DNA replication with its complex structure [
1]. So, clustered DNA damage is thought to be strongly involved in the biological effectiveness of heavy ion beam. However, a lot of studies have presented no certain correlation between yields of clustered DNA damage and severity of radiation effect. We previously indicated that the yields of clustered DNA damage decreased with increasing LET in the DNA molecules irradiated in test tubes with gamma rays, and carbon and iron ion beams whose showed different LET, respectively [
2]. In this study, we aimed to reveal correlation between clustered DNA damage and the LET of heavy ion beam in the irradiated cells.
In the experiments, Chinese hamster ovary AA8 cells growing exponentially were irradiated by carbon, silicon, argon and iron ion beams from Heavy Ion Medical Accelerator in Chiba (HIMAC) of the National Institute of Radiological Sciences, Japan. These LETs were 13, 55, 90 and 200 keV/µm, respectively. For comparison, we used gamma rays from 137Cs-gamma source, Gammacell 40 (Atomic Energy of Canada Ltd), at Saga University. The irradiated cells were subjected by static-field gel electrophoresis to quantify clustered DNA damage of the genomic DNA. For this analysis, we used Fpg and endonuclease III for clustered DNA damage including oxidative purine and pyrimidine lesions, respectively. We also analysed the corresponding isolated DNA damages by aldehyde reactive probe method [
3], and the surviving fractions of the irradiated cells in this study.
The electrophoretic results indicated that total yields of clustered DNA damage in the irradiated cells decreased with increasing LET, including the double-strand break (DSB) and the respective clustered base damages (Fig. 1). This result conforms to our previous study with the irradiated DNA molecules [
2]. The damage kinetics is thought to be mainly derived from two reasons: decreasing fluxes and increasing reaction with reactive oxygen species each other in increase in LET. In the clustered DNA damage induced by each radiation, the most decremental fraction was clustered base damage, but not DSB. The isolated DNA damages decreased with increasing LET like clustered DNA damage in this study (data not shown). These results make us realize the degree of contribution of direct and indirect effects of ionizing radiation. The certain amount of DSB were derived from the direct effect and showed less reactivity to LET. In contrast, oxidative base lesions were mainly generated by indirect effect with reactive oxygen species, which sensitively responded to LET change. We also found seemingly conflicted result of the relationship between LET and RBE (data not shown). We need further study to elucidate act of clustered DNA damage in radiobiological effect with heavy ion beams.
Fig. 1.The yields of clustered DNA damages in the cells irradiated with respective ionizing radiations. Each clustered DNA damage consists of DSB (open bar) and clustered base damage (closed bar), and calculated from the strength of released band on electrophoretic gel.
Clinical trial registration number if required: None.
heavy ion beam; clustered DNA damage; LET; RBE
Telomeres distinguish chromosome ends from double-strand breaks (DSBs) and prevent chromosome fusion. However, telomeres can also interfere with DNA repair, as shown by a deficiency in nonhomologous end joining (NHEJ) and an increase in large deletions at telomeric DSBs. The sensitivity of telomeric regions to DSBs is important in the cellular response to ionizing radiation and oncogene-induced replication stress, either by preventing cell division in normal cells, or by promoting chromosome instability in cancer cells. We have previously proposed that the telomeric protein TRF2 causes the sensitivity of telomeric regions to DSBs, either through its inhibition of ATM, or by promoting the processing of DSBs as though they are telomeres, which is independent of ATM. Our current study addresses the mechanism responsible for the deficiency in repair of DSBs near telomeres by combining assays for large deletions, NHEJ, small deletions, and gross chromosome rearrangements (GCRs) to compare the types of events resulting from DSBs at interstitial and telomeric DSBs. Our results confirm the sensitivity of telomeric regions to DSBs by demonstrating that the frequency of GCRs is greatly increased at DSBs near telomeres and that the role of ATM in DSB repair is very different at interstitial and telomeric DSBs. Unlike at interstitial DSBs, a deficiency in ATM decreases NHEJ and small deletions at telomeric DSBs, while it increases large deletions. These results strongly suggest that ATM is functional near telomeres and is involved in end protection at telomeric DSBs, but is not required for the extensive resection at telomeric DSBs. The results support our model in which the deficiency in DSB repair near telomeres is a result of ATM-independent processing of DSBs as though they are telomeres, leading to extensive resection, telomere loss, and GCRs involving alternative NHEJ.
The ends of chromosomes, called telomeres, prevent chromosome ends from appearing as DNA double-strand breaks (DSBs) and prevent chromosome fusion by forming a specialized nucleo-protein complex. The critical function of telomeres in end protection has a downside, in that it interferes with the repair of DSBs that occur near telomeres. DSBs are critical DNA lesions, because if they are not repaired correctly they can result in gross chromosome rearrangements (GCRs). As a result, the deficiency in DSB repair near telomeres has now been implicated in ageing, by promoting cell senescence, and cancer, by promoting telomere dysfunction due to oncogene-induced replication stress. The studies presented here demonstrate that DSBs near telomeres commonly result in GCRs in a human tumor cell line. Moreover, our results demonstrate that the mechanism of repair of telomeric DSBs is very different from the mechanism of repair of DSBs at other locations, supporting our hypothesis that the deficiency in repair of DSBs near telomeres is a result of the abnormal processing of DSBs due to the presence of telomeric proteins. Understanding the mechanism responsible for the deficiency in DSB repair near telomeres will provide important insights into critical human disease pathways.
Various types of radiation effects in mammalian cells have been studied with the aim to predict the radiosensitivity of tumours and normal tissues, e.g. DNA double strand breaks (DSB), chromosome aberrations and cell reproductive inactivation. However, variation in correlations with clinical results has reduced general application. An additional type of information is required for the increasing application of high-LET radiation in cancer therapy: the Relative Biological Effectiveness (RBE) for effects in tumours and normal tissues. Relevant information on RBE values might be derived from studies on cells in culture.
To evaluate relationships between DNA-DSB, chromosome aberrations and the clinically most relevant effect of cell reproductive death, for ionizing radiations of different LET, dose-effect relationships were determined for the induction of these effects in cultured SW-1573 cells irradiated with gamma-rays from a Cs-137 source or with α-particles from an Am-241 source. RBE values were derived for these effects. Ionizing radiation induced foci (IRIF) of DNA repair related proteins, indicative of DSB, were assessed by counting gamma-H2AX foci. Chromosome aberration frequencies were determined by scoring fragments and translocations using premature chromosome condensation. Cell survival was measured by colony formation assay. Analysis of dose-effect relations was based on the linear-quadratic model.
Our results show that, although both investigated radiation types induce similar numbers of IRIF per absorbed dose, only a small fraction of the DSB induced by the low-LET gamma-rays result in chromosome rearrangements and cell reproductive death, while this fraction is considerably enhanced for the high-LET alpha-radiation. Calculated RBE values derived for the linear components of dose-effect relations for gamma-H2AX foci, cell reproductive death, chromosome fragments and colour junctions are 1.0 ± 0.3, 14.7 ± 5.1, 15.3 ± 5.9 and 13.3 ± 6.0 respectively.
These results indicate that RBE values for IRIF (DNA-DSB) induction provide little valid information on other biologically-relevant end points in cells exposed to high-LET radiations. Furthermore, the RBE values for the induction of the two types of chromosome aberrations are similar to those established for cell reproductive death. This suggests that assays of these aberrations might yield relevant information on the biological effectiveness in high-LET radiotherapy.
DNA double-strand breaks (DSBs), which are formed by the Spo11 protein, initiate meiotic recombination. Previous DSB-mapping studies have used rad50S or sae2Δ mutants, which are defective in break processing, to accumulate Spo11-linked DSBs, and report large (≥ 50 kb) “DSB-hot” regions that are separated by “DSB-cold” domains of similar size. Substantial recombination occurs in some DSB-cold regions, suggesting that DSB patterns are not normal in rad50S or sae2Δ mutants. We therefore developed a novel method to map genome-wide, single-strand DNA (ssDNA)–associated DSBs that accumulate in processing-capable, repair-defective dmc1Δ and dmc1Δ rad51Δ mutants. DSBs were observed at known hot spots, but also in most previously identified “DSB-cold” regions, including near centromeres and telomeres. Although approximately 40% of the genome is DSB-cold in rad50S mutants, analysis of meiotic ssDNA from dmc1Δ shows that most of these regions have substantial DSB activity. Southern blot assays of DSBs in selected regions in dmc1Δ, rad50S, and wild-type cells confirm these findings. Thus, DSBs are distributed much more uniformly than was previously believed. Comparisons of DSB signals in dmc1, dmc1 rad51, and dmc1 spo11 mutant strains identify Dmc1 as a critical strand-exchange activity genome-wide, and confirm previous conclusions that Spo11-induced lesions initiate all meiotic recombination.
During meiosis, the two copies of each chromosome present in the full (diploid) genome come together and then separate, forming haploid gametes (sperm and eggs, in animals). Recombination, which swaps DNA between chromosomes, is critical for chromosome pairing and separation, and also promotes genetic diversity in the next generation, providing the feedstock for evolution. DNA double-strand breaks (DSBs), which are formed by the conserved Spo11 nuclease, initiate meiotic recombination. DSB mapping is thus an alternative to standard genetic analysis for determining where meiotic recombination occurs. DSBs have been most extensively mapped in budding yeast mutants that fail to remove Spo11 from break ends, blocking further recombination steps. Paradoxically, those studies indicated that DSBs are absent from large regions where recombination was known to occur. We developed a new DSB mapping method that purifies and analyzes the single-strand DNA formed at breaks after Spo11 removal. This new map shows that DSBs (and by inference, recombination) actually occur frequently throughout almost all of the budding yeast genome, in a distribution that is consistent with recombination's roles in chromosome pairing and in generating genetic diversity. This new mapping method will be useful for studying meiotic recombination and DNA damage repair in other organisms.
The authors developed a new method to detect DNA damage genome-wide, and they used it to show that meiotic recombination is more uniformly distributed in budding yeast than was previously believed.
Purpose: Particle beams are increasingly applied to various cancer treatments because of their excellent dose localization to tumors while preserving surrounding normal tissues. However, characteristic of DNA damages induced by particle beams and their repair mechanisms are not fully understood. It is known that the majority of DNA double-strand breaks (DSBs) induced by ionizing radiation are repaired either by non-homologous end-joining (NHEJ) or by homologous recombination (HR) pathways. However, it has not been clarified how NHEJ and HR pathways contribute to the repair of DSBs induced by various particle beams [1, 2]. Thus, the purpose of this study is to clarify how these repair pathways contribute to the DSB repair in cells after irradiation with various radiation qualities.
Material and methods: A control Chinese hamster ovary (CHO) cell line (AA8), its mutant cell line deficient of DNA-PKcs (V3), XRCC4 (XR1) and Chinese hamster lung fibroblast cell line deficient of XRCC2 (IRS1) were exposed to gamma rays, protons, carbon ions and iron ions. V3 and XR1 lack NHEJ pathway, while IRS1 lacks HR pathway. After each irradiation, colony survival and gross-chromosome aberration were examined.
Results: It was demonstrated that colony survival was clearly dependent on the presence of NHEJ or HR pathways as well as radiation qualities. Although HR-deficient cells (IRS1) became more sensitive as LET value increased, NHEJ-deficient cells (V3 and XR1) did not further sensitized as LET value increased (Fig.
1). In addition, values of relative biological effectiveness of iron beams were higher in HR-deficient cells than in NHEJ-deficient cells (3.2 in AA8; 2.7 in IRS1; 1.8 in XR1 and V3). These may suggest that HR plays an important role in repairing DNA lesions induced by high-LET radiation. As for the incidence of total chromosomal aberration, we found that its incidence increased as LET values increased in wild-type (AA8) and NHEJ-deficient cells (V3, XR1), but not in HR-deficient cells (IRS1) (Table
1). However, occurrence of chromosome-type aberrations increased as LET values increased in all cell lines analysed here. This may indicate that the chromosomal aberrations occur from not only unrepaired damages but also the repair process of error-prone NHEJ pathway, suggesting that limited capacity of NHEJ to repair DSBs induced by high-LET irradiation may cause increased number of chromosome-type aberrations.
Conclusions: Taken together, although NHEJ pathway is the major pathway to repair DSBs induced by various types of radiation, HR pathway may play more important roles as LET value increases.
Fig. 1.Radio sensitivity after gamma ray and iron beam. Clonogenic survival curves of AA8 (closed circle); XR1 (open circle); V3 (closed square) and IRS1 (open square) after irradiation with gamma ray (dashed) and iron beam (dotted). NHEJ-deficient cells are more sensitive to gamma ray, but HR-deficient cells are most sensitive to iron beam.
Table 1.Chromatid and chromosome type aberration per chromosomeCell linesTypeControlProtonCarbonIronAA8 (wild-type)Chromatid0.860.240.611.07Chromosome0.051.180.743.44XR1 (NHEJ)Chromatid0.053.181.553.41Chromosome02.592.504.96V3 (NHEJ)Chromatid0.483.372.417.89Chromosome0.165.264.978.01IRS1 (HR)Chromatid0.028.2610.357.59Chromosome0.014.247.438.18
high LET particle; NHEJ and HR pathway; chromosome aberration
Cancer treatment with high LET heavy ion beam, especially, carbon ion beam (12C), is becoming very popular over conventional radiotherapy like low LET gamma or X-ray. Combination of Poly(ADP-ribose) polymerase (PARP) inhibitor with xenotoxic drugs or conventional radiation (gamma or X-ray) is the newer approach for cancer therapy. The aim of our study was to compare the radiosensitivity and induction of apoptosis by high LET 12C and low LET gamma radiation in HeLa and PARP-1 knocked down cells. We did comet assay to detect DNA breaks, clonogenic survival assay, and cell cycle analysis to measure recovery after DNA damage. We measured apoptotic parameters like nuclear fragmentation and caspase-3 activation. DNA damage, cell killing, and induction of apoptosis were significantly higher for 12C than gamma radiation in HeLa. Cell killing and apoptosis were further elevated upon knocking down of PARP-1. Both 12C and gamma induced G2/M arrest although the 12C had greater effect. Unlike the gamma, 12C irradiation affects DNA replication as detected by S-phase delay in cell cycle analysis. So, we conclude that high LET 12C has greater potential over low LET gamma radiation in killing cells and radiosensitization upon PARP-1 inhibition was several folds greater for 12C than gamma.
Hexavalent chromium (Cr(VI)) is a widespread environmental contaminant and a known human carcinogen, generally causing bronchial cancer. Recent studies have shown that the particulate forms of Cr(VI) are the potent carcinogens. Particulate Cr(VI) is known to induce a spectrum of DNA damage such as DNA single strand breaks, Cr-DNA adducts, DNA-protein crosslinks and chromosomal aberrations. However, particulate Cr(VI)-induced DNA double strand breaks (DSBs) have not been reported. Thus, the aim of this study was to determine if particulate Cr(VI)-induces DSBs in human bronchial cells. Using the single cell gel electrophoresis assay (comet assay), showed that lead chromate-induced concentration dependent increases in DSBs with 0.1, 0.5, 1 and 5 μg/cm2 lead chromate inducing a 20, 50, 67 and 109% relative increase in the tail integrated intensity ratio, respectively. Sodium chromate at concentrations of 1, 2.5 and 5 μM induced 38, 78 and 107% relative increase in the tail integrated intensity ratio, respectively. We also show that genotoxic concentrations of lead chromate activate the ataxia telangiectasia mutated (ATM) protein, which is thought to play a central role in the early stages of DSB detection and controls cellular responses to this damage. The H2A.X protein becomes rapidly phosphorylated on residue serine 139 in cells when DSBs are introduced into the DNA by ionizing radiation. By using immunofluorescence, we found that lead chromate-induced concentration-dependent increases in phosphorylated H2A.X (r-H2A.X) foci formation with 0.1, 0.5, 1, 5 and 10 μg/cm2 lead chromate inducing a relative increase in the number of cells with r-H2A.X foci formation of 43, 51, 115 and 129%, respectively.
Hexavalent chromium; DNA double strand breaks; ATM; Smc1; Lead chromate
Etoposide is a cancer drug that induces strand breaks in cellular DNA by inhibiting topoisomerase II (topoII) religation of cleaved DNA molecules. Although DNA cleavage by topoisomerase II always produces topoisomerase II-linked DNA double-strand breaks (DSBs), the action of etoposide also results in single-strand breaks (SSBs), since religation of the two strands are independently inhibited by etoposide. In addition, recent studies indicate that topoisomerase II-linked DSBs remain undetected unless topoisomerase II is removed to produce free DSBs.
To examine etoposide-induced DNA damage in more detail we compared the relative amount of SSBs and DSBs, survival and H2AX phosphorylation in cells treated with etoposide or calicheamicin, a drug that produces free DSBs and SSBs. With this combination of methods we found that only 3% of the DNA strand breaks induced by etoposide were DSBs. By comparing the level of DSBs, H2AX phosphorylation and toxicity induced by etoposide and calicheamicin, we found that only 10% of etoposide-induced DSBs resulted in histone H2AX phosphorylation and toxicity. There was a close match between toxicity and histone H2AX phosphorylation for calicheamicin and etoposide suggesting that the few etoposide-induced DSBs that activated H2AX phosphorylation were responsible for toxicity.
These results show that only 0.3% of all strand breaks produced by etoposide activate H2AX phosphorylation and suggests that over 99% of the etoposide induced DNA damage does not contribute to its toxicity.
Double-strand breaks (DSBs) are highly deleterious DNA lesions as they lead to chromosome aberrations and/or apoptosis. The formation of nuclear DSBs triggers phosphorylation of histone H2AX on Ser-139 (defined as γH2AX), which participates in the repair of such DNA damage. Our aim was to compare the induction of γH2AX in relation to DSBs induced by topoisomerase II (TOPO II) poisons, etoposide (ETOP) and mitoxantrone (MXT), in V79 cells. DSBs were measured by the neutral comet assay, while γH2AX was quantified using immunocytochemistry and flow cytometry. Stabilized cleavage complexes (SCCs), lesions thought to be responsible for TOPO II poison-induced genotoxicity, were measured using a complex of enzyme–DNA assay. In the case of ETOP, a no observed adverse effect level (NOAEL) and lowest observed effect level (LOEL) for genotoxicity was determined; γH2AX levels paralleled DSBs at all concentrations but significant DNA damage was not detected below 0.5 μg/ml. Furthermore, DNA damage was dependent on the formation of SCCs. In contrast, at low MXT concentrations (0.0001–0.001 μg/ml), induction of γH2AX was not accompanied by increases in DSBs. Rather, DSBs were only significantly increased when SCCs were detected. These findings suggest MXT-induced genotoxicity occurred via at least two mechanisms, possibly related to DNA intercalation and/or redox cycling as well as TOPO II inhibition. Our findings also indicate that γH2AX can be induced by DNA lesions other than DSBs. In conclusion, γH2AX, when measured using immunocytochemical and flow cytometric methods, is a sensitive indicator of DNA damage and may be a useful tool in genetic toxicology screens. ETOP data are consistent with the threshold concept for TOPO II poison-induced genotoxicity and this should be considered in the safety assessment of chemicals displaying an affinity for TOPO II and genotoxic/clastogenic effects.
DNA double-strand breaks; Topoisomerase II; Etoposide; Mitoxantrone; Neutral comet assay; γH2AX; Genotoxicity thresholds
The performance of adult stem cells is crucial for tissue homeostasis but their regenerative capacity declines with age, leading to failure of multiple organs. In skeletal muscle this failure is manifested by the loss of functional tissue, the accumulation of fibrosis, and reduced satellite cell-mediated myogenesis in response to injury. While recent studies have shown that changes in the composition of the satellite cell niche are at least in part responsible for the impaired function observed with aging, little is known about the effects of aging on the intrinsic properties of satellite cells. For instance, their ability to repair DNA damage and the effects of a potential accumulation of DNA double strand breaks (DSBs) on their regenerative performance remain unclear. This work demonstrates that old muscle stem cells display no significant accumulation of DNA DSBs when compared to those of young, as assayed after cell isolation and in tissue sections, either in uninjured muscle or at multiple time points after injury. Additionally, there is no significant difference in the expression of DNA DSB repair proteins or globally assayed DNA damage response genes, suggesting that not only DNA DSBs, but also other types of DNA damage, do not significantly mark aged muscle stem cells. Satellite cells from DNA DSB-repair-deficient SCID mice do have an unsurprisingly higher level of innate DNA DSBs and a weakened recovery from gamma-radiation-induced DNA damage. Interestingly, they are as myogenic in vitro and in vivo as satellite cells from young wild type mice, suggesting that the inefficiency in DNA DSB repair does not directly correlate with the ability to regenerate muscle after injury. Overall, our findings suggest that a DNA DSB-repair deficiency is unlikely to be a key factor in the decline in muscle regeneration observed upon aging.
In cells exposed to low linear energy transfer (LET) ionizing-radiation (IR),
double-strand-breaks (DSBs) form within clustered-damage-sites (CDSs) from
lesions disrupting the DNA sugar-phosphate backbone. It is commonly assumed that
all DSBs form promptly and are immediately detected by the cellular
DNA-damage-response (DDR) apparatus. However, there is evidence that the pool of
DSBs detected by physical methods, such as pulsed-field gel electrophoresis
(PFGE), comprises not only promptly forming DSBs (prDSBs) but also DSBs
developing during lysis at high temperatures from thermally-labile sugar-lesions
(TLSLs). We recently demonstrated that conversion of TLSLs to DNA breaks and
ultimately to DSBs also occurs in cells during the first hour of
post-irradiation incubation at physiological temperatures. Thus, TLSL-dependent
DSBs (tlDSBs) are not an avoidable technique-related artifact, but a reality the
cell always faces. The biological consequences of tlDSBs and the dependence of
their formation on LET require in-depth investigation. Heavy-ions (HI) are a
promising high-LET radiation modality used in cancer treatment. HI are also
encountered in space and generate serious radiation protection problems to
prolonged space missions. Here, we study, therefore, the effect of HI on the
yields of tlDSBs and prDSBs. We report a reduction in the yield of tlDBSs
stronger than that earlier reported for neutrons, and with pronounced cell line
dependence. We conclude that with increasing LET the complexity of CDSs
increases resulting in a commensurate increase in the yield prDSBs and a
decrease in tlDSBs. The consequences of these effects to the relative biological
effectiveness are discussed.
DNA double strand breaks (DSB); Ionizing radiation (IR); High LET; Heavy ions; Labile lesions; Radiation chemistry
Plants exhibit a robust transcriptional response to gamma radiation which includes the induction of transcripts required for homologous recombination and the suppression of transcripts that promote cell cycle progression. Various DNA damaging agents induce different spectra of DNA damage as well as “collateral” damage to other cellular components and therefore are not expected to provoke identical responses by the cell. Here we study the effects of two different types of ionizing radiation (IR) treatment, HZE (1 GeV Fe26+ high mass, high charge, and high energy relativistic particles) and gamma photons, on the transcriptome of Arabidopsis thaliana seedlings. Both types of IR induce small clusters of radicals that can result in the formation of double strand breaks (DSBs), but HZE also produces linear arrays of extremely clustered damage. We performed these experiments across a range of time points (1.5–24 h after irradiation) in both wild-type plants and in mutants defective in the DSB-sensing protein kinase ATM. The two types of IR exhibit a shared double strand break-repair-related damage response, although they differ slightly in the timing, degree, and ATM-dependence of the response. The ATM-dependent, DNA metabolism-related transcripts of the “DSB response” were also induced by other DNA damaging agents, but were not induced by conventional stresses. Both Gamma and HZE irradiation induced, at 24 h post-irradiation, ATM-dependent transcripts associated with a variety of conventional stresses; these were overrepresented for pathogen response, rather than DNA metabolism. In contrast, only HZE-irradiated plants, at 1.5 h after irradiation, exhibited an additional and very extensive transcriptional response, shared with plants experiencing “extended night.” This response was not apparent in gamma-irradiated plants.
DNA repair; double-strand breaks; transcriptomics; stress; cell-cycle; ionizing radiation; HZE; gamma radiation
Regardless of the achievable remissions with first line hormone therapy in patients with prostate cancer (CaP), the disease escapes the hormone dependent stage to a more aggressive status where chemotherapy is the only effective treatment and no treatment is curative. This makes it very important to identify new targets that can improve the outcome of treatment. ATM and DNA-PK are the two kinases responsible for signalling and repairing double strand breaks (DSB). Thus, both kinases are pertinent targets in CaP treatment to enhance the activity of the numerous DNA DSB inducing agents used in CaP treatment such as ionizing radiation (IR). Colony formation assay was used to assess the sensitivity of hormone dependent, p53 wt (LNCaP) and hormone independent p53 mutant (PC3) CaP cell lines to the cytotoxic effect of IR and Doxorubicin in the presence or absence of Ku55933 and NU7441 which are small molecule inhibitors of ATM and DNA-PK, respectively. Flow cytometry based methods were used to assess the effect of the two inhibitors on cell cycle, apoptosis and H2AX foci formation. Neutral comet assay was used to assess the induction of DNA DSBs. Ku55933 or NU7441 alone increased the sensitivity of CaP cell lines to the DNA damaging agents, however combining both inhibitors together resulted in further enhancement of sensitivity. The cell cycle profile of both cell lines was altered with increased cell death, DNA DSBs and H2AX foci formation. This study justifies further evaluation of the ATM and DNA-PK inhibitors for clinical application in CaP patients. Additionally, the augmented effect resulting from combining both inhibitors may have a significant implication for the treatment of CaP patients who have a defect in one of the two DSB repair pathways.
Bacterial Dsb enzymes are involved in the oxidative folding of many proteins, through the formation of disulfide bonds between their cysteine residues. The Dsb protein network has been well characterized in cells of the model microorganism Escherichia coli. To gain insight into the functioning of the Dsb system in epsilon-Proteobacteria, where it plays an important role in the colonization process, we studied two homologs of the main Escherichia coli Dsb oxidase (EcDsbA) that are present in the cells of the enteric pathogen Campylobacter jejuni, the most frequently reported bacterial cause of human enteritis in the world.
Methods and Results
Phylogenetic analysis suggests the horizontal transfer of the epsilon-Proteobacterial DsbAs from a common ancestor to gamma-Proteobacteria, which then gave rise to the DsbL lineage. Phenotype and enzymatic assays suggest that the two C. jejuni DsbAs play different roles in bacterial cells and have divergent substrate spectra. CjDsbA1 is essential for the motility and autoagglutination phenotypes, while CjDsbA2 has no impact on those processes. CjDsbA1 plays a critical role in the oxidative folding that ensures the activity of alkaline phosphatase CjPhoX, whereas CjDsbA2 is crucial for the activity of arylsulfotransferase CjAstA, encoded within the dsbA2-dsbB-astA operon.
Our results show that CjDsbA1 is the primary thiol-oxidoreductase affecting life processes associated with bacterial spread and host colonization, as well as ensuring the oxidative folding of particular protein substrates. In contrast, CjDsbA2 activity does not affect the same processes and so far its oxidative folding activity has been demonstrated for one substrate, arylsulfotransferase CjAstA. The results suggest the cooperation between CjDsbA2 and CjDsbB. In the case of the CjDsbA1, this cooperation is not exclusive and there is probably another protein to be identified in C. jejuni cells that acts to re-oxidize CjDsbA1. Altogether the data presented here constitute the considerable insight to the Epsilonproteobacterial Dsb systems, which have been poorly understood so far.
Class-specific plaque-forming cell (PFC) (gammaM, gamma1, gamma2, and gammaA) responses to type III pneumococcal polysaccharide (SSS-III) were studied in BALB/c x C57BL/6F1 (CBF1) mice with and without induction of an allogeneic effect. Gamma1, gamma2, and gammaA PFC were detected in two ways: (a) With the sequential development of the assay slides, first for direct (gammaM)PFC followed by incubation with class- specific antiimmunoglobulin and complement for the development of additional gamma1, gamma2, and gammaA PFC (gammaM-independent gamma1, gamma2, and gammaA PFC); and (b) by blocking gammaM PFC with goat anti- gammaM and simultaneously developing gamma1, gamma2, and gammaA PFC (total gamma1-, gamma2-, and gammaA-secreting PFC). The results showed that whereas gammaM PFC arose on the 3rd d after immunization, gamma1-, gamma2-, and gammaA-secreting PFC arose on the 4th to 5th d after immunization. They appeared in association with gammaM-secreting PFC because they were detected with the gammaM blocking method but not with the sequential method. By the 7th d most gamma1, gamma2, and gammaA PFC were detected by the sequential method as well, indicating that those antibodies were secreted independently of cells secreting gammaM. When the numbers of double-class-secreting PFC were evaluated on the 5th d, the following results were obtained: 83% of gammaM PFC were secreting either gamma1 (25%), gamma2 (55%), or gammaA (2%). We interpret these data as evidence for an antigen-driven class differentiation from gammaM to gammaA and from gammaM to gammaG in the majority of anti-SSS- III-secreting clones without T-cell help. When an allogeneic effect was provided by inoculation of parental BALB/c spleen cells together with antigen, the numbers of all classes of PFC were increased. Furthermore, the frequency of gammaM-gammaG (108%) or gammaM-gammaA (9%) double- class secretors was increased, and gammaM-independent gammaG and gammaA secretors were detected earlier, indicating an overall maturation- promoting effect. In addition, prolonged appearance of gammaA PFC was dependent on the allogeneic effect.
Reviewed are the methods aimed to detect DNA damage in individual cells, estimate its extent and relate it to cell cycle phase and induction of apoptosis. They include the assays that reveal DNA fragmentation during apoptosis, as well as DNA damage induced by genotoxic agents. DNA fragmentation that occurs in the course of apoptosis is detected by selective extraction of degraded DNA. DNA in chromatin of apoptotic cells shows also increased propensity to undergo denaturation. The most common assay of DNA fragmentation relies on labelling DNA strand breaks with fluorochrome-tagged deoxy-nucleotides. The induction of double-strand DNA breaks (DSBs) by genotoxic agents provides a signal for histone H2AX phosphorylation on Ser139; the phosphorylated H2AX is named γH2AX. Also, ATM-kinase is activated through its autophosphorylation on Ser1981. Immunocytochemical detection of γH2AX and/or ATM-Ser1981(P) are sensitive probes to reveal induction of DSBs. When used concurrently with analysis of cellular DNA content and caspase-3 activation, they allow one to correlate the extent of DNA damage with the cell cycle phase and with activation of the apoptotic pathway. The presented data reveal cell cycle phase-specific patterns of H2AX phosphorylation and ATM autophosphorylation in response to induction of DSBs by ionizing radiation, topoisomerase I and II inhibitors and carcinogens. Detection of DNA damage in tumour cells during radio-or chemotherapy may provide an early marker predictive of response to treatment.
Immune responses and DNA damage repair are two fundamental processes that have been characterized extensively, but the links between them remain largely unknown. We report that multiple bacterial, fungal and oomycete plant pathogen species induce double-strand breaks (DSBs) in host plant DNA. DNA damage detected by histone γ-H2AX abundance or DNA comet assays arose hours before the disease-associated necrosis caused by virulent Pseudomonas syringae pv. tomato. Necrosis-inducing paraquat did not cause detectable DSBs at similar stages after application. Non-pathogenic E. coli and Pseudomonas fluorescens bacteria also did not induce DSBs. Elevation of reactive oxygen species (ROS) is common during plant immune responses, ROS are known DNA damaging agents, and the infection-induced host ROS burst has been implicated as a cause of host DNA damage in animal studies. However, we found that DSB formation in Arabidopsis in response to P. syringae infection still occurs in the absence of the infection-associated oxidative burst mediated by AtrbohD and AtrbohF. Plant MAMP receptor stimulation or application of defense-activating salicylic acid or jasmonic acid failed to induce a detectable level of DSBs in the absence of introduced pathogens, further suggesting that pathogen activities beyond host defense activation cause infection-induced DNA damage. The abundance of infection-induced DSBs was reduced by salicylic acid and NPR1-mediated defenses, and by certain R gene-mediated defenses. Infection-induced formation of γ-H2AX still occurred in Arabidopsis atr/atm double mutants, suggesting the presence of an alternative mediator of pathogen-induced H2AX phosphorylation. In summary, pathogenic microorganisms can induce plant DNA damage. Plant defense mechanisms help to suppress rather than promote this damage, thereby contributing to the maintenance of genome integrity in somatic tissues.
Multicellular organisms are continuously exposed to microbes and have developed sophisticated defense mechanisms to counter attack by microbial pathogens. Organisms also encounter many types of DNA damage and have evolved multiple mechanisms to maintain their genomic integrity. Even though these two fundamental responses have been characterized extensively, the relationship between them remains largely unclear. Our study demonstrates that microbial plant pathogens with diverse life styles, including bacteria, oomycete and fungal pathogens, induce double-strand breaks (DSBs) in the genomes of infected host plant cells. DSB induction is apparently a common feature during plant-pathogen interactions. DSBs are the most deleterious form of DNA damage and can lead to chromosomal aberrations and gene mutations. In response to pathogen infection, plant immune responses are activated and contribute to suppressing pathogen-induced DSBs, thereby maintaining better genome integrity and stability. The findings identify important ways that the plant immune and DNA damage repair responses are interconnected. Awareness of the above phenomena may foster future development of disease management approaches that improve crop productivity under biotic stress.