Damage clusters after gamma irradiation of a nanoparticulate plasmid DNA peptide condensate

We have gamma-irradiated plasmid DNA in aqueous solution in the presence of submillimolar concentrations of the ligand tetra-arginine. Depending upon the ionic strength, under these conditions, the plasmid can adopt a highly compacted and aggregated form which attenuates by some two orders of magnitude the yield of damage produced by the indirect effect. The yields of DNA single- and double-strand breaks (SSB and DSB) which result are closely comparable with those produced in living cells. The radical lifetimes, diffusion distances, and track structure are expected to be similarly well reproduced. After irradiation, the aggregation was reversed by adjusting the ionic conditions. The approximate spatial distribution of the resulting DNA damage was then assayed by comparing the increases in the SSB and DSB yields produced by a subsequent incubation with limiting concentrations of the eukaryotic base excision repair enzymes formamidopyrimidine-DNA N-glycosylase (the FPG protein) and endonuclease III. Smaller increases in DSB yields were observed in the plasmid target that was irradiated in the condensed form. By modeling the spatial distribution of DNA damage, this result can be interpreted in terms of a greater extent of damage clustering.     

Radiosensitization of DNA by gold nanoparticles irradiated with high-energy electrons

Zheng, Y., Hunting, D. J., Ayotte, P. and Sanche, L. Radiosensitization of DNA by Gold Nanoparticles Irradiated with High-Energy Electrons. Radiat. Res. 168, 19-27 (2008). Thin films of pGEM-3Zf(-) plasmid DNA were bombarded by 60 keV electrons with and without gold nanoparticles. DNA single- and double-strand breaks (SSBs and DSBs) were measured by agarose gel electrophoresis. From transmission electron micrographs, the gold nanoparticles were found to be closely linked to DNA scaffolds, probably as a result of electrostatic binding. The probabilities for formation of SSBs and DSBs from exposure of 1:1 and 2:1 gold nanoparticle:plasmid mixtures to fast electrons increase by a factor of about 2.5 compared to neat DNA samples. For monolayer DNA adsorbed on a thick gold substrate, the damage increases by an order of magnitude. The results suggest that the enhancement of radiosensitivity is due to the production of additional low-energy secondary electrons caused by the increased absorption of ionizing radiation energy by the metal, in the form of gold nanoparticles or of a thick gold substrate. Since short-range low-energy secondary electrons are produced in large amounts by any type of ionizing radiation, and since on average only one gold nanoparticle per DNA molecule is needed to increase damage considerably, targeting the DNA of cancer cells with gold nanoparticles may offer a novel approach that is generally applicable to radiotherapy treatments.     

Mechanisms underlying production of double-strand breaks in plasmid DNA after decay of 125I-Hoechst

Previously, the kinetics of strand break production by (125)I-labeled m-iodo-p-ethoxyHoechst 33342 ((125)IEH) in supercoiled (SC) plasmid DNA had demonstrated that approximately 1 DSB is produced per (125)I decay both in the presence and absence of the hydroxyl radical scavenger DMSO. In these experiments, an (125)IEH:DNA molar ratio of 42:1 was used. We now hypothesize that this DSB yield (but not the SSB yield) may be an overestimate due to subsequent decays occurring in any of the 41 (125)IEH molecules still bound to nicked (N) DNA. To test our hypothesis, (125)IEH was incubated with SC pUC19 plasmids ((125)IEH:DNA ratio of approximately 3:1) and the SSB and DSB yields were quantified after the decay of (125)I. As predicted, the number of DSBs produced per (125)I decay is one-half that reported previously ( approximately 0.5 compared to approximately 1, +/- DMSO) whereas the number of SSBs ( approximately 3/(125)I decay) is similar to that obtained previously ( approximately 90% are generated by OH radicals). Direct visualization by atomic force microscopy confirms formation of L and N DNA after (125)IEH decays in SC DNA and supports the strand break yields reported. These findings indicate that although SSB production is independent of the number of (125)IEH bound to DNA, the DSB yield can be augmented erroneously by (125)I decays occurring in N DNA. Further analysis indicates that 17% of SSBs and 100% of DSBs take place within the plasmid molecule in which an (125)IEH molecule decays, whereas 83% of SSBs are formed in neighboring plasmid DNA molecules.     

Reaction rate coefficients of hydroxyl radical-induced DNA single- and α-type double-strand breaks

With a model system of pBR322 plasmid DNA solution in vitro, the dose effects of radiation- induced single- and double-strand breaks (SSB and DSB) were measured and DSB was distinguished into α- and β-types. Under the condition of low scavenging capacity existing in the irradiated DNA solution, SSB and αDSB were mainly induced by hydroxyl radicals (^·OH). Moreover, a certain relationship was obtained between the SSB and αDSB yields and the DNA concentration. It was found that when the DNA solution was irradiated in the presence of 2.5 mmol dm^–3 mannitol, the reciprocals of G(SSB) and G(αDSB), respectively, were linearly related to the reciprocal of the DNA concentration, i.e. the competition reactions of DNA and mannitol for ^·OH radicals can be described by second-order kinetics. The rate coefficients and the efficiencies of the ^·OH radical inducing SSB were deduced. Also, the reaction rate coefficients and the efficiencies for the induction of αDSB from SSB by the ^·OH radical transfer mechanism, were first derived from the competition kinetics. Received: 27 October 1999 / Accepted: 15 March 2000     

Induction of DNA damage, including abasic sites, in plasmid DNA by carbon ion and X-ray irradiation

DNA from plasmid pUC18 was irradiated with low-LET (13 keV/μm) or high-LET (60 keV/μm) carbon ions or X-rays (4 keV/μm) in solutions containing several concentrations of Tris (0.66–200 mM) to determine the yield of abasic (AP) sites and the effect of scavenging capacity. The yield of AP sites, detected as single-strand breaks (SSB) after digestion with E. coli endonuclease IV (Nfo), was compared with that of SSB and base lesions. At higher concentrations of Tris, the yields of single or clustered AP sites were significantly lower than those of single or clustered base lesions. The relative yields of single AP sites and AP clusters were less than 10 and 7 %, respectively, of the total damage produced at a scavenger capacity mimicking that in cells. The dependence of the yield of AP sites on scavenging capacity was similar to that of prompt strand breaks. The ratios of the yield of isolated AP sites to that of SSB induced by carbon ion or X-ray irradiation were relatively constant at 0.45 ± 0.15 over the tested range of scavenger capacity, although the ratio of SSB to double-strand breaks (DSB) showed the characteristic dependence on both scavenging capacity and radiation quality. These results indicate that the reaction of water radiolysis products, presumably OH radicals, with the sugar-phosphate moieties in the DNA backbone induces both AP sites and SSB with similar efficiency. Direct ionization of DNA is notably more involved in the production of DSB and base lesion clusters than in the production of AP site clusters.     

Clustered DNA damage induced by gamma radiation in human fibroblasts (HF19), hamster (V79-4) cells and plasmid DNA is revealed as Fpg and Nth sensitive sites

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.     

Influence of radiation quality on the yield of DNA strand breaks in SV40 DNA irradiated in solution.

Using highly energetic particles to irradiate plasmid DNA in aerobic aqueous solution, we have compiled an extensive database on how yields of DNA single- and double-strand breaks (SSBs and DSBs) vary with radiation quality. This study was performed in a low-scavenging buffer system and covers a wide range of ion species (helium to uranium) and LETs (5 to 16,000 keV/microm). For LETs up to around 40 keV/microm for SSBs and 400 keV/microm for DSBs, the total energy deposition determines cross section. At higher LET, cross sections level off and individual plateaus for particles of different atomic numbers are observed. For each ion species this is more pronounced and occurs at lower LET for SSBs than for DSBs, leading to an increase in the DSB:SSB ratio from 1:70 for X rays to 1:6 at 500 keV/microm. At this LET, the influence of track structure becomes evident, with high local concentrations of ionization events favoring the formation of DSBs and also intratrack recombination reactions. For lower-energy ions, a saturation in production of measurable DSBs is apparent, due to correlated lesion induction within densely ionizing particle tracks. For very heavy low-energy ions, both SSB and DSB cross sections decrease with particle velocity at nearly constant LET, forming individual hooked curves when plotted as a function of LET.     

Investigation on the correlation between energy deposition and clustered DNA damage induced by low-energy electrons

This study presents the correlation between energy deposition and clustered DNA damage, based on a Monte Carlo simulation of the spectrum of direct DNA damage induced by low-energy electrons including the dissociative electron attachment. Clustered DNA damage is classified as simple and complex in terms of the combination of single-strand breaks (SSBs) or double-strand breaks (DSBs) and adjacent base damage (BD). The results show that the energy depositions associated with about 90% of total clustered DNA damage are below 150 eV. The simple clustered DNA damage, which is constituted of the combination of SSBs and adjacent BD, is dominant, accounting for 90% of all clustered DNA damage, and the spectra of the energy depositions correlating with them are similar for different primary energies. One type of simple clustered DNA damage is the combination of a SSB and 1–5 BD, which is denoted as SSB?+?BD. The average contribution of SSB?+?BD to total simple clustered DNA damage reaches up to about 84% for the considered primary energies. In all forms of SSB?+?BD, the SSB?+?BD including only one base damage is dominant (above 80%). In addition, for the considered primary energies, there is no obvious difference between the average energy depositions for a fixed complexity of SSB?+?BD determined by the number of base damage, but average energy depositions increase with the complexity of SSB?+?BD. In the complex clustered DNA damage constituted by the combination of DSBs and BD around them, a relatively simple type is a DSB combining adjacent BD, marked as DSB?+?BD, and it is of substantial contribution (on average up to about 82%). The spectrum of DSB?+?BD is given mainly by the DSB in combination with different numbers of base damage, from 1 to 5. For the considered primary energies, the DSB combined with only one base damage contributes about 83% of total DSB?+?BD, and the average energy deposition is about 106 eV. However, the energy deposition increases with the complexity of clustered DNA damage, and therefore, the clustered DNA damage with high complexity still needs to be considered in the study of radiation biological effects, in spite of their small contributions to all clustered DNA damage.     

Calculation of radiation-induced DNA damage from photons and tritium beta-particles

Radiation-induced damage in nucleosomal DNA was modelled by Monte Carlo means. An atomistic representation of DNA with a first hydration shell was used. DNA single- and double-strand break (SSB and DSB) yields were calculated for ¹³⁷Cs photons, x-rays and tritium beta-particles. Monte Carlo-generated electron tracks for liquid water were used to model energy deposition. Chemical evolution of a track and interactions between species and DNA following water radiolysis were modelled in an encounter-controlled manner. The effects of varying the scavenging capacity of the environment, the extent of DNA protection by histones and the setting of a threshold for direct energy depositions on DNA break yields were all systematically studied. DSB complexity was assessed in terms of DNA breaks and base damage accompanying a DSB. Model parameters were adjusted to make predictions consistent with experimental data on DSB yields and yield modification by a variety of factors including changing DNA conformation and incorporation of scavengers. An embedded model of nucleosomal DNA by histones was required to explain experimentally observed modification of DSB yield by removal of bound histones. Complex DSB, defined as DSB accompanied by an additional strand breakage, exhibited high association with base damage. It is shown that hydroxyl radical interactions with bases are a major contributor to DSB complexity. On average there were 1.15 and 2.69 OH-base interactions accompanying simple and complex DSB, respectively for ¹³⁷Cs. Over 80% of complex DSB had at least one OH-base interaction associated with a DNA break. Received: 21 March 2000 / Accepted: 27 October 2000     

Cleavage of cellular DNA by calicheamicin gamma1

It is assumed that the efficient antitumor activity of calicheamicin gamma1 is mediated by its ability to introduce DNA double-strand breaks in cellular DNA. To test this assumption we have compared calicheamicin gamma1-mediated cleavage of cellular DNA and purified plasmid DNA. Cleavage of purified plasmid DNA was not inhibited by excess tRNA or protein indicating that calicheamicin gamma1 specifically targets DNA. Cleavage of plasmid DNA was not affected by incubation temperature. In contrast, cleavage of cellular DNA was 45-fold less efficient at 0 degrees C as compared to 37 degrees due to poor cell permeability at low temperatures. The ratio of DNA double-strand breaks (DSB) to single-stranded breaks (SSB) in cellular DNA was 1:3, close to the 1:2 ratio observed when calicheamicin gamma1 cleaved purified plasmid DNA. DNA strand breaks introduced by calicheamicin gamma1 were evenly distributed in the cell population as measured by the comet assay. Calicheamicin gamma1-induced DSBs were repaired slowly but completely and resulted in high levels of H2AX phosphorylation and efficient cell cycle arrest. In addition, the DSB-repair deficient cell line Mo59J was hyper sensitive to calicheamicin gamma. The data indicate that DSBs is the crucial damage after calicheamicin gamma1 and that calicheamicin gamma1-induced DSBs are recognized normally. The high DSB:SSB ratio, specificity for DNA and the even damage distribution makes calicheamicin gamma1 a superior drug for studies of the DSB-response and emphasizes its usefulness in treatment of malignant disease.     

Monte Carlo simulation of strand-break induction on plasmid DNA in aqueous solution by monoenergetic electrons

The radiation-induced process of strand breaks on pBR322 plasmid DNA in aqueous solution for different energy electrons was studied by Monte Carlo simulation. Assumptions of induction mechanisms of single- and double-strand breaks (SSBs and DSBs) used in the simulation are that SSB is induced by OH or H reaction with DNA and that DSB is induced by two SSBs on the opposite strands within 10 bp. Dose-response relationships of SSBs and DSBs were demonstrated for monoenergetic electrons of 100 eV, 10 keV, 1 keV and 1 MeV, and the yields of SSB and DSB were calculated. The dose-response relationships of SSBs and DSBs can be fitted by linear and linear-quadratic functions, respectively. The ratio of quadratic to linear components of DSB induction changes due to the electron energy. A high contribution of the linear component is observed for 1 keV electrons in the dose range below 160 Gy. The yields of SSBs and DSBs for all examined electron energies lie well within the experimental data when the probability of strand-break induction by OH and H is assumed to be around 0.1-0.2. The yield of SSBs has a minimum at 1 keV, while the yield of DSBs has a maximum at 1 keV in the examined energies. The strand breaks are formed most densely for 1 keV electrons.     

Relative contribution of homologous recombination and non-homologous end-joining to DNA double-strand break repair after oxidative stress in Saccharomyces cerevisiae

Oxidative damage to DNA seems to be an important factor in developing many human diseases including cancer. It involves base and sugar damage, base-free sites, DNA-protein cross-links and DNA single-strand (SSB) and double-strand (DSB) breaks. Oxidative DSB can be formed in various ways such as their direct induction by the drug or their generation either through attempted and aborted repair of primary DNA lesions or through DNA replication-dependent conversion of SSB. In general, two main pathways are responsible for repairing DSB, homologous recombination (HR) and non-homologous end-joining (NHEJ), with both of them being potential candidates for the repair of oxidative DSB. We have examined relative contribution of HR and NHEJ to cellular response after oxidative stress in Saccharomyces cerevisiae. Therefore, cell survival, mutagenesis and DSB induction and repair in the rad52, yku70 and rad52 yku70 mutants after hydrogen peroxide (H(2)O(2)), menadione (MD) or bleomycin (BLM) exposure were compared to those obtained for the corresponding wild type. We show that MD exposure does not lead to observable DSB induction in yeast, suggesting that the toxic effects of this agent are mediated by other types of DNA damage. Although H(2)O(2) treatment generates some DSB, their yield is relatively low and hence DSB may only partially be responsible for toxicity of H(2)O(2), particularly at high doses of the agent. On the other hand, the basis of the BLM toxicity resides primarily in DSB induction. Both HR and NHEJ act on BLM-induced DSB, although their relative participation in the process is not equal. Based on our results we suggest that the complexity and/or the quality of the BLM-induced DSB might represent an obstacle for the NHEJ pathway.     

Novel method for quantifying radiation-induced single-strand-break yields in plasmid DNA highlights 10-fold discrepancy

The widely used agarose gel electrophoresis method for assessing radiation-induced single-strand-break (SSB) yield in plasmid DNA involves measurement of the fraction of relaxed-circular (C) form that migrates independently from the intact supercoiled (SC) form. We rationalized that this method may underestimate the SSB yield since the position of the relaxed-circular form is not altered when the number of SSB per DNA molecule is >1. To overcome this limitation, we have developed a novel method that directly probes and quantifies SSBs. Supercoiled ³H-pUC19 plasmid samples were irradiated with γ-rays, alkali-denatured, dephosphorylated, and kinated with γ-[³²P]ATP, and the DNA-incorporated ³²P activities were used to quantify the SSB yields per DNA molecule, employing a standard curve generated using DNA molecules containing a known number of SSBs. The same irradiated samples were analyzed by agarose gel and SSB yields were determined by conventional methods. Comparison of the data demonstrated that the mean SSB yield per plasmid DNA molecule of [21.2 ± 0.59] × 10⁻² Gy⁻¹ as measured by direct probing is ∼10-fold higher than that obtained from conventional gel-based methods. These findings imply that the SSB yields inferred from agarose gels need reevaluation, especially when they were utilized in the determination of radiation risk.     

99mTc Pyrene Derivative Complex Causes Double-Strand Breaks in dsDNA Mainly through Cluster-Mediated Indirect Effect in Aqueous Solution

Radiation therapy for cancer patients works by ionizing damage to nuclear DNA, primarily by creating double-strand breaks (DSB). A major shortcoming of traditional radiation therapy is the set of side effect associated with its long-range interaction with nearby tissues. Low-energy Auger electrons have the advantage of an extremely short effective range, minimizing damage to healthy tissue. Consequently, the isotope ^99mTc, an Auger electron source, is currently being studied for its beneficial potential in cancer treatment. We examined the dose effect of a pyrene derivative ^99mTc complex on plasmid DNA by using gel electrophoresis in both aqueous and methanol solutions. In aqueous solutions, the average yield per decay for double-strand breaks is 0.011±0.005 at low dose range, decreasing to 0.0005±0.0003 in the presence of 1 M dimethyl sulfoxide (DMSO). The apparent yield per decay for single-strand breaks (SSB) is 0.04±0.02, decreasing to approximately a fifth with 1 M DMSO. In methanol, the average yield per decay of DSB is 0.54±0.06 and drops to undetectable levels in 2 M DMSO. The SSB yield per decay is 7.2±0.2, changing to 0.4±0.2 in the presence of 2 M DMSO. The 95% decrease in the yield of DSB in DMSO indicates that the main mechanism for DSB formation is through indirect effect, possibly by cooperative binding or clustering of intercalators. In the presence of non-radioactive ligands at a near saturation concentration, where radioactive Tc compounds do not form large clusters, the yield of SSB stays the same while the yield of DSB decreases to the value in DMSO. DSBs generated by ^99mTc conjugated to intercalators are primarily caused by indirect effects through clustering.     

DNA damage in peripheral blood lymphocytes in patients during combined chemotherapy for breast cancer

Combined chemotherapy is used for the treatment of a number of malignancies such as breast cancer. The target of these antineoplastic agents is nuclear DNA, although it is not restricted to malignant cells. The aim of the present study was to assess DNA damage in peripheral blood lymphocytes (PBLs) of breast cancer patients subjected to combined adjuvant chemotherapy (5-fluorouracil, epirubicin and cyclophosphamide, FEC), using a modified comet assay to detect DNA single-strand breaks (SSB) and double-strand breaks (DSB).

Forty-one female patients with advanced breast cancer before and after chemotherapy and 60 healthy females participated in the study. Alkaline and neutral comet assays were performed in PBLs according to a standard protocol, and DNA tail moment was measured by a computer-based image analysis system.

Breast cancer patients before treatment had higher increased background levels of SSB and DSB as compared to healthy women. During treatment, a significant increase in DNA damage was observed after the 2nd cycle, which persisted until the end of treatment. Eighty days after the end of treatment the percentage of PBLs with SSB and DSB remained elevated, but the magnitude of DNA damage (tail moment) returned to baseline levels. There was no correlation between PBL DNA damage and response to chemotherapy.

DNA-SSB and DSB in PBLs are present in cancer patients before treatment and increase significantly after combined chemotherapy. No correlation with response to adjuvant chemotherapy was found. Biomonitoring DNA damage in PBLs of cancer patients could help prevent secondary effects and the potential risks of developing secondary cancers.     

Induction and rejoining of gamma-ray-induced DNA single- and double-strand breaks in Chinese hamster AA8 cells and in two radiosensitive clones

The induction and rejoining of gamma-ray-induced DNA strand breaks were measured in a Chinese hamster ovary cell line, AA8, and in two radiosensitive clones (EM9 and NM2) derived from it. The kinetics of recovery from sublethal damage (SLD) and potentially lethal damage (PLD) has previously been characterized in each of these lines [vanAnkeren et al., Radiat. Res., 115, 223-237 (1988)]. No significant differences were observed among the cell lines in the yields of either DNA single-strand breaks (SSBs) or double-strand breaks (DSBs) as assayed by filter elution. Data for SSB rejoining in AA8 and NM2 cells irradiated with 7.5 Gy were fit by a biexponential process (t1/2 values of approximately 4 and 80 min). In comparison, SSB rejoining in EM9 cells was initially slower (t1/2 = 10 min) and a higher level of SSBs was unrejoined 6 h after irradiation. DSB rejoining in AA8 cells assayed at pH 9.6 was also biphasic (t1/2 values of 15 and 93 min), although when assayed at pH 7.0, most (approximately 80%) of the damage was rejoined at a constant rate (t1/2 = 45 min) during the first 2 h. EM9 cells exhibited a slower initial rate of DSB rejoining when assayed at pH 9.6 but showed no difference compared with AA8 cells in DSB rejoining when assayed at pH 7.0. These results indicate that radiosensitive EM9 cells, whose kinetics of recovery from SLD and PLD was the same as that of AA8 cells, have a defect in the fast phase of SSB rejoining but no measurable defect in DSB rejoining. Conversely, NM2 cells, which displayed a reduced shoulder width on their survival curve and decreased recovery from SLD, had no demonstrable defects in the rate or extent of rejoining of DSBs or SSBs. When compared with the SLD and PLD data reported previously, these results suggest that there is no direct correlation between either of these recovery processes and the rejoining of SSBs or DSBs as assayed here.     

Further studies of the induction and intracellular repair of DNA strand breaks using intranuclear SV40 as a test system

We have previously published the techniques and preliminary results of an SV40 viral probe assay for gamma-radiation-induced single- and double-strand DNA breaks and their intracellular repair in higher cells (Radiat. Res. 101, 356-372, 1985). Those experiments with SV40 infected CV-1 monkey kidney cells suggested that this assay technique demonstrates slow but extensive intracellular repair of single-strand breaks (SSB), and possible early repair of double-strand breaks (DSB), followed by later induction of DSB. Following up on these early observations, many additional infection-incubation experiments have now been performed with both human and simian cells. Analysis of data from these experiments involving up to 6 h of postinfection intranuclear incubation shows the same distribution of strand break damage in incubated and unincubated samples. This implies that under these experimental conditions there is neither intracellular repair nor further production of SSB or DSB in intranuclear viral DNA. We have evidence which suggests that this lack of repair or degradation occurs because the bulk of intranuclear SV40 DNA is relatively inaccessible to host cell enzymes.     

Condensin I interacts with the PARP-1-XRCC1 complex and functions in DNA single-strand break repair

Condensins are essential protein complexes critical for mitotic chromosome organization. Little is known about the function of condensins during interphase, particularly in mammalian cells. Here we report the interphase-specific interaction between condensin I and the DNA nick-sensor poly(ADP-ribose) polymerase 1 (PARP-1). We show that the association between condensin I, PARP-1, and the base excision repair (BER) factor XRCC1 increases dramatically upon single-strand break damage (SSB) induction. Damage-specific association of condensin I with the BER factors flap endonuclease 1 (FEN-1) and DNA polymerase delta/epsilon was also observed, suggesting that condensin I is recruited to interact with BER factors at damage sites. Consistent with this, DNA damage rapidly stimulates the chromatin association of PARP-1, condensin I, and XRCC1. Furthermore, depletion of condensin in vivo compromises SSB but not double-strand break (DSB) repair. Our results identify a SSB-specific response of condensin I through PARP-1 and demonstrate a role for condensin in SSB repair.     

Zebularine induces replication-dependent double-strand breaks which are preferentially repaired by homologous recombination

Zebularine is a second-generation, highly stable hydrophilic inhibitor of DNA methylation with oral bioavailability that preferentially target cancer cells. It acts primarily as a trap for DNA methyl transferases (DNMTs) protein by forming covalent complexes between DNMT protein and zebularine-substrate DNA. It’s well documented that replication-blocking DNA lesions can cause replication fork collapse and thereby to the formation of DNA double-strand breaks (DSB). DSB are dangerous lesions that can lead to potentially oncogenic genomic rearrangements or cell death. The two major pathways for repair of DSB are non-homologous end joining (NHEJ) and homologous recombination (HR). Recently, multiple functions for the HR machinery have been identified at arrested forks. Here we investigate in more detail the importance of the lesions induced by zebularine in terms of DNA damage and cytotoxicity as well as the role of HR in the repair of these lesions. When we examined the contribution of NHEJ and HR in the repair of DSB induced by zebularine we found that these breaks were preferentially repaired by HR. Also we show that the production of DSB is dependent on active replication. To test this, we determined chromosome damage by zebularine while transiently inhibiting DNA synthesis. Here we report that cells deficient in single-strand break (SSB) repair are hypersensitive to zebularine. We have observed more DSB induced by zebularine in XRCC1 deficient cells, likely to be the result of conversion of SSB into toxic DSB when encountered by a replication fork. Furthermore we demonstrate that HR is required for the repair of these breaks. Overall, our data suggest that zebularine induces replication-dependent DSB which are preferentially repaired by HR.     

Calculation of radiation-induced DNA damage from photons and tritium beta-particles

Yields of DNA single- and double-strand breaks (SSB and DSB) in nucleosomal DNA were calculated for 137Cs, 70 keV photons and tritium beta-particles by Monte Carlo means. Monte Carlo-generated electron tracks for liquid water were used to model energy deposition. Chemical evolution of a track and interactions between species and DNA following water radiolysis were modelled in an encounter-controlled manner. The calculated relative biological effectiveness (RBE) for DSB production for tritium against 137Cs was 1.2 for the total DSB yield. Tritium beta-particles were slightly more efficient compared to 137Cs in producing complex DSB, defined as DSB accompanied by additional strand breaks. The RBE for complex DSB formation was 1.3. Most complex DSB exhibited associated base damage; the extent of the base damage was similar for all the radiation types considered. Correlated DSB conforming to nucleosome periodicity were observed. However, their frequency was low, of the order of 2% of total DSB. For all the DNA damage endpoints considered and their response to variation of the scavenging environment or DNA conformation no difference was observed between 70 keV photons and tritium beta-particles.