Clustered DNA Damage Research Articles

Nanoscale analysis of clustered DNA damage after high-LET irradiation by quantitative electron microscopy--the heavy burden to repair.

Low- and high-linear energy transfer (LET) ionising radiation are effective cancer therapies, but produce structurally different forms of DNA damage. Isolated DNA damage is repaired efficiently; however, clustered lesions may be more difficult to repair, and are considered as significant biological endpoints. We investigated the formation and repair of DNA double-strand breaks (DSBs) and clustered lesions in human fibroblasts after exposure to sparsely (low-LET; delivered by photons) and densely (high-LET; delivered by carbon ions) ionising radiation. DNA repair factors (pKu70, 53BP1, γH2AX, and pXRCC1) were detected using immunogold-labelling and electron microscopy, and spatiotemporal DNA damage patterns were analysed within the nuclear ultrastructure at the nanoscale level. By labelling activated Ku-heterodimers (pKu70) the number of DSBs was determined in electron-lucent euchromatin and electron-dense heterochromatin. Directly after low-LET exposure (5 min post-irradiation), single pKu70 dimers, which reflect isolated DSBs, were randomly distributed throughout the entire nucleus with a linear dose correlation up to 30 Gy. Most euchromatic DSBs were sensed and repaired within 40 min, whereas heterochromatic DSBs were processed with slower kinetics. Essentially all DNA lesions induced by low-LET irradiation were efficiently rejoined within 24h post-irradiation. High-LET irradiation caused localised energy deposition within the particle tracks, and generated highly clustered DNA lesions with multiple DSBs in close proximity. The dimensions of these clustered lesions along the particle trajectories depended on the chromatin packing density, with huge DSB clusters predominantly localised in condensed heterochromatin. High-LET irradiation-induced clearly higher DSB yields than low-LET irradiation, with up to ∼ 500 DSBs per μm(3) track volume, and large fractions of these heterochromatic DSBs remained unrepaired. Hence, the spacing and quantity of DSBs in clustered lesions influence DNA repair efficiency, and may determine the radiobiological outcome.

Localization estimation of ionizing radiation-induced abasic sites in DNA in the solid state using fluorescence resonance energy transfer.

Clustered DNA damage is considered an important factor in determining the biological consequences of ionizing radiation. In this study, we successfully estimated the localization of abasic sites (APs) in DNA exposed to ionizing radiation using fluorescence resonance energy transfer (FRET) without any involvement of repair enzyme functions. A linearized plasmid (pUC19 digested by Sma I) was irradiated with: (60)Co γ rays; (4)He(2+) (2.0 MeV/u) particles; and the (12)C(5+) (0.37 MeV/u) particles in the solid state. A donor or acceptor fluorescent probe with a nucleophilic O-amino group was used to label APs. The results showed that the (12)C(5+) particle likely produced close APs within a track. The apparent distance calculated from the observed FRET efficiency (E) of around 0.10 was estimated to be approximately 17 base pairs. On the other hand, E values of (60)Co γ rays and the (4)He(2+) beam were less than those of the (12)C(5+) beam, increased with increasing AP density (the average number of APs per base pair), and were slightly greater than those of randomly distributed APs. We propose that the FRET method provides a degree of localization regardless of whether an AP cluster is single-stranded or bistranded DNA damage.

Clustered DNA damage on subcellular level: effect of scavengers.

Clustered DNA damages are induced by ionizing radiation, particularly of high linear energy transfer (LET). Compared to isolated DNA damage sites, their biological effects can be more severe. We investigated a clustered DNA damage induced by high LET radiation (C 290 MeV u(-1) and Fe 500 MeV u(-1)) in pBR322 plasmid DNA. The plasmid is dissolved in pure water or in aqueous solution of one of the three scavengers (coumarin-3-carboxylic acid, dimethylsulfoxide, and glycylglycine). The yield of double strand breaks (DSB) induced in the DNA plasmid-scavenger system by heavy ion radiation was found to decrease with increasing scavenging capacity due to reaction with hydroxyl radical, linearly with high correlation coefficients. The yield of non-DSB clusters was found to occur twice as much as the DSB. Their decrease with increasing scavenging capacity had lower linear correlation coefficients. This indicates that the yield of non-DSB clusters depends on more factors, which are likely connected to the chemical properties of individual scavengers.

Direct observation of preferential processing of clustered abasic DNA damages with APE1 in TATA box and CpG island by reaction kinetics and fluorescence dynamics

Sequences like the core element of TATA box and CpG island are frequently encountered in the genome and related to transcription. The fate of repair of clustered abasic sites in such sequences of genomic importance is largely unknown. This prompted us to investigate the sequence dependence of cleavage efficiency of APE1 enzyme at abasic sites within the core sequences of TATA box and CpG island using fluorescence dynamics and reaction kinetics. Simultaneous molecular dynamics study through steady state and time resolved fluorescence spectroscopy using unique ethidium bromide dye release assay confirmed an elevated amount of abasic site cleavage of the TATA box sequence as compared to the core CpG island. Reaction kinetics showed that catalytic efficiency of APE1 for abasic site cleavage of core CpG island sequence was ∼4 times lower as compared to that of the TATA box. Higher value of Km was obtained from the core CpG island sequence than the TATA box sequence. This suggests a greater binding effect of APE1 enzyme on TATA sequence that signifies a prominent role of the sequence context of the DNA substrate. Evidently, a faster response from APE1 was obtained for clustered abasic damage repair of TATA box core sequences than CpG island consensus sequences. The neighboring bases of the abasic sites in the complementary DNA strand were found to have significant contribution in addition to the flanking bases in modulating APE1 activity. The repair refractivity of the bistranded clustered abasic sites arise from the slow processing of the second abasic site, consequently resulting in decreased overall production of potentially lethal double strand breaks.

SU-E-T-645: Dose Enhancement to Cell Nucleus Due to Hard Collisions of Protons with Electrons in Gold Nanospheres

Purpose: The purpose of this study was to investigate the theoretical dose enhancement to a cell nucleus due to increased fluence of secondary electrons when gold nanospheres are present in the cytoplasm during proton therapy. Methods: We modeled the irradiation of prostate cancer cells using protons of variable energies when 10,000 gold nanoparticles, each with radius of 10 nm, were randomly distributed in the cytoplasm. Using simple analytical equations, we calculated the increased mean dose to the cell nucleus due to secondary electrons produced by hard collisions of 0.1, 1, 10, and 100 MeV protons with orbital electrons in gold. We only counted electrons with kinetic energy higher than 1 keV. In addition to calculating the increase in the mean dose to the cell nucleus, we also calculated the increase in local dose in the “shadow,” i.e., the umbra, of individual gold nanospheres due to forward scattered electrons. Results: For proton energies of 0.1, 1, 10, and 100 MeV, we calculated increases to the mean nuclear dose of 0.15, 0.09, 0.05, and 0.04%, respectively. When we considered local dose increases in the shadows of individual gold spheres, we calculated local dose increases of 5.5, 3.2, 1.9, and 1.3%, respectively. Conclusion:more » We found negligible, less than 0.2%, increases in the mean dose to the cell nucleus due to electrons produced by hard collisions of protons with electrons in gold nanospheres. However, we observed increases up to 5.5% in the local dose in the shadow of gold nanospheres. Considering the shadow radius of 10 nm, these local dose enhancements may have implications for slightly increased probability of clustered DNA damage when gold nanoparticles are close to the nuclear membrane.« less

A novel approach to study radiation track structure with nanometer-equivalent resolution

Clustered DNA damages are considered the critical lesions in the pathways leading from the initial energy deposition by radiation to radiobiological damage. The spatial distribution of the initial DNA damage is mainly determined by radiation track-structure at the nanometer level. In this work, a novel experimental approach to image the three-dimensional structure of micrometric radiation track segments is presented. The approach utilizes the detection of single ions created in low-pressure gas. Ions produced by radiation drift towards a GEM-like 2D hole-pattern detector. When entering individual holes, ions can induce ion-impact ionization of the working-gas starting a confined electron avalanche that generates the output signal. By registering positive ions rather than electrons, diffusion is reduced and a spatial resolution of the track image of the order of water-equivalent nanometers can be achieved. Measurements and simulations to characterize the performance of a few detector designs were performed. Different cathode materials were tested and ionization cluster size distributions of 241Am alpha particles were measured. The electric field configuration in the detector was calculated to optimize the ion focusing into the detector holes. The preliminary results obtained show the directions for further development of the detector.

Non-DSB clustered DNA lesions induced by ionizing radiation are largely responsible for the loss of plasmid DNA functionality in the presence of cisplatin

The combination of cisplatin and ionizing radiation (IR) increases cell toxicity by both enhancing DNA damage and inhibiting repair mechanisms. Although the formation of cluster DNA lesions, particularly double-strand breaks (DSB) at the site of cisplatin–DNA-adducts has been reported to induce cell death, the contribution of DSB and non-DSB cluster lesions to the cellular toxicity is still unknown. Although both lesions are toxic, it is not always possible to measure their frequency and cell survival in the same model system. To overcome this problem, here, we investigate the effect of cisplatin-adducts on the induction of DSB and non-DSB cluster DNA lesions by IR and determine the impact of such lesions on plasmid functionality. Cluster lesions are two or more lesions on opposite DNA strands with a short distance such that error free repair is difficult or impossible. At a ratio of two cisplatin per plasmid, irradiation of platinated DNA in solution with 137Cs γ-rays shows enhancements in the formation of DNA DSB and non-DSB cluster lesions by factors of 2.6 and 2.1, respectively, compared to unmodified DNA. However, in absolute terms, the yield for non-DSB cluster lesions is far larger than that for DSB, by a factor of 26. Unmodified and cisplatin-modified DNA were irradiated and subsequently transformed into Escherichia coli to give survival curves representing the functionality of the plasmid DNA as a function of radiation dose. Our results demonstrate that non-DSB cluster lesions are the only toxic lesions present at a sufficient frequency to account for the loss of DNA functionality. Our data also show that Frank-DSB lesions are simply too infrequent to account for the loss of DNA functionality. In conclusion, non-DSB cluster DNA damage is known to be difficult to repair and is probably the lesion responsible for the loss of functionality of DNA modified by cisplatin.

Fast Monte Carlo simulation of DNA damage induction by Auger-electron emission

Purpose: A local damage model (LDM) was developed to estimate the biological efficiency of Auger-electron-emitting radionuclides.Materials and methods: The LDM required information on the local dose distribution, local energy spectrum, and clustered DNA damage yields in the cell nucleus. To apply the model, the nucleus was divided into concentric shells where each shell contributed its own local dose, energy spectrum, and damage yield. The local doses and energy spectra were computed using the PENELOPE (PENetration and Energy LOss of Positrons and Electrons) code. The DNA damage yields were estimated using the MCDS (Monte Carlo damage simulation) code.Results: For a typical 4-μm radius mammalian cell nucleus, the absorbed doses to the cell nucleus per unit cumulated activity, equal to 0.0065, 0.00418, 0.0028, 0.0027 and 0.0015 Gy Bq−1 s−1 for 125I, 119Sb, 123I, 111In and 99mTc, were within 6% difference with the MIRD (Medical Internal Radiation Dose) published data. The simulated total (simple and complex) single-strand break (SSB) and double-strand break (DSB) yields were in the same order, i.e., 125I > 119Sb > 123I > 111In > 99mTc. The agreement between present results and literature data for the DNA damage yields was generally good. More than 75% of the total SSB and DSB yields were contributed from regions within 2.5 μm of the nucleus center.Conclusions: The proposed methodology was computationally efficient and could be applied to other irradiation geometries such as cell clusters.

Quantitative characteristics of clustered DNA damage in irradiated cells by heavy ion beams

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.

Order effect of repair processes to clustered DNA damage

In a living cell, a multiply damaged site in DNA is thought to be processed by several different pathways simultaneously or sequentially. Under this situation, the cellular response to the lesion cluster might depend on the order of repair processes because the configuration of the lesions will be modified by the reaction of initial repair protein, affecting the DNA-binding or lesion-excision activities of latter repair proteins. For example, a cluster comprised an AP site or SSB and base lesions is formed after one of the lesions in a base lesion cluster is excised by a glycosylase. It has been predicted that the enzymatic activity of a protein binding to a lesion could be affected by a second lesion located nearby. In the present study, we investigate whether initial base excision repair process affects secondary process on the clustered DNA damage site produced by high linear energy transfer irradiation.Plasmid DNA (pUC18) used as model DNA were irradiated in a solution (10 mM Tris and 1 mM EDTA) with C6+ ions (60 keV/µm) obtained from HIMAC (NIRS, Chiba, Japan) according to our previous method [ 1], or He2+ ions from TIARA (JAEA, Takasaki, Japan) in a fully hydrated film [ 2]. We used two base excision repair enzymes to examine the order effect of BER processes, Nth and Fpg, which convert pyrimidine and purine lesions to an SSB, respectively. After exposure, the sample was incubated with Nth (or Fpg) at 37°C for 30 min. Then, we added another enzyme Fpg (or Nth) and incubate for another 30 min. We also simultaneously treated the DNA sample with both enzymes for 1 h. After the incubation, the samples were analysed by agarose (1%) gel electrophoresis to determine the yield of conformational changes of the plasmid from closed circular form to open or linear form. These changes show productions of an enzymatically induced SSB (the former) or DSB (the latter).A typical example of the dependence of the loss of closed circular form DNA on radiation dose is shown in Fig. 1a for carbon ion irradiation. The reciprocal of the slope of each graph shows yield of prompt SSBs [n(SSB)prompt] for the sample without any enzymatic treatment, or [n(SSB)prompt] + enzymatically induced SSBs [n(SSB)Nth(or Fpg, or Nth+Fpg)]. The SSB yield for the sample treated with Nth first and then Fpg is very slightly less than that of other treatments (<10%). The dose responses of the linear form DNA production are shown in Fig. 1b. There were no significant differences among the enzymatic treatments. These results indicate that the configuration change of the cluster by the first enzymatic treatment does not significantly influence the activity of secondary enzyme. The most of base lesion clusters produced in the track of carbon ions might be converted to strand breaks, some of which form double-strand breaks (DSBs), by glycosylase activity in living cells. Fig. 1.Dependence of (a) the loss of closed-circular DNA and (b) the number of linear form DNA DSB determined from the fraction of the linear form of DNA, on carbon ion dose. The arrows indicate the order of the enzymatic treatments.

Dependence of the yields of AP sites and AP clusters produced in plasmid DNA on scavenging capacity and LET

An apurinic or apyrimidinic site (AP site) has known to be one of the typical DNA lesions induced by ionizing irradiation to cells. A clustered DNA damage site composed of AP sites (AP clusters) can be visualized as a double-strand break (DSB) by a treatment of DNA sample with endonuclease IV proteins as an enzymatic probe. AP clusters are efficiently induced in human cells by low LET γ- and X-irradiation [ 1] with the similar yields with those for the clusters which contain pyrimidine or purine base lesions. However, there has been very little knowledge of the mechanistic aspects of the production of AP sites and AP clusters. Recently, we reported the dependence of the yields of AP sites and AP clusters, as well as base lesions, strand breaks and base lesion clusters induced by irradiation of carbon ions (LET: 13 and 60 keV/μm) obtained from HIMAC (NIRS, Chiba, Japan) on radical scavenging capacity in the DNA samples [ 2].In the present study, we have performed theoretical calculation for the production of AP sites as well as those of strand breaks and base lesions by the carbon ion exposure (13 keV/μm). A Monte Carlo track structure simulation code for ion tracks, TRACION, was used for this work according to our previous study [ 3]. As a DNA model molecule, simple linear DNA with 150 bp was applied for the calculation. Several scavenging capacities of the samples are tested to estimate the effect of indirect action of diffusible OH radicals in the damage induction in the experimental conditions.We assumed that the AP site formation pathway originates from a common intermediate of an OH radical adduct with that of single-strand breaks (SSBs) or base lesions (Scheme 1). In order to reproduce the experimentally obtained yields of AP sites, we newly determined the branching ratio of induction of AP site to that of SSBs or base lesions to be 17:33 or 2:48, respectively, in the simulation. The calculated yields of AP site, as well as SSB, were well consistent with those for experimentally obtained (Fig. 1). However, the calculated yields for AP clusters were one-fifth of those for experimental data. The yields for DSBs in lower scavenging capacities were also one-fifth of those for experimental ones in a lower scavenging capacity, thought they were well consistent with each other above a cell mimetic condition (>3 × 108 s). In order to bridge a gap between theoretical and experimental yields, we need a novel mechanism of damage-clustering such as dissociative low energy electron attachment or hole-migration onto a DNA molecule. Fig. 1.Dependence of the yield of AP site or AP cluster (A) and prompt SSB or prompt DSB (B) on scavenging capacity in the sample. The experimental data below a scavenging capacity of 109 s are cited from [ 2] and those at 1010 s in (B) are from [ 4]. Scheme 1.Reactions of DNA with an OH radical and the branching ratios among the damage induction pathways.

Effects of (5'S)-5',8-cyclo-2'-deoxyadenosine on the base excision repair of oxidatively generated clustered DNA damage. A biochemical and theoretical study.

The presence of 5',8-cyclo-2'-deoxyadenosine (5'S)-cdA induces modifications in the geometry of the DNA duplex in the 5'-end direction of the strand and in the 3'-end direction of the complementary strand. As a consequence, the enzymes are probably not able to adjust their active sites in this rigid structure. Additionally, clustered DNA damage sites, a signature of ionising radiation, pose a severe challenge to a cell's repair machinery, particularly base excision repair (BER). To date, clusters containing a DNA base lesion, (5'S)-cdA, which is repaired by nucleotide excision repair, have not been explored. We have therefore investigated whether bistranded clusters containing (5'S)-cdA influence the repairability of an opposed AP site lesion, which is repaired by BER. Using synthetic oligonucleotides containing a bistranded cluster with (5'S)-cdA and an AP site at different interlesion separations, we have shown that in the presence of (5'S)-cdA on the 5'-end side, repair of the AP site by the BER machinery is retarded when the AP site is ≤8 bases from the (5'S)-cdA. However, if (5'S)-cdA is located on the 3'-end side with respect to the AP site, the effect on its repair is much weaker and totally disappears for distances ≥8 bases.

The Relative Contributions of DNA Strand Breaks, Base Damage and Clustered Lesions to the Loss of DNA Functionality Induced by Ionizing Radiation

The majority of studies on lethal radiobiological damage have focused on double-strand breaks (DSBs), a type of clustered DNA damage and the evaluation of their toxicity, while other types of clustered DNA damage have received much less attention. The main purpose of this study is to evaluate the contribution of different lesions induced by ionizing radiation to the loss of plasmid DNA functionality. We employed a simple model system comprising E. coli transformed with an irradiated plasmid [pGEM-3Zf (-)] to determine the effect of DSBs and other lesions including base damage and clustered lesions on the functionality ("viability") of the plasmid. The yields of γ-radiation-induced single-strand breaks (SSBs) and DSBs were measured by gel electrophoresis. We found that the transformation efficiency decreases with radiation dose, but this decrease cannot be explained by the formation of DSBs. For example, at doses of 500 and 700 Gy, the relative transformation efficiency falls from 100% to 53% and 26%, respectively, while only 5.7% and 9.1% of the plasmids contain a DSB. In addition, it is also unlikely that randomly distributed base lesions could explain the loss of functionality of the plasmid, since cells can repair them efficiently. However, clustered lesions other than DSBs, which are difficult to repair and result in the loss of information on both DNA strands, have the potential to induce the loss of plasmid functionality. We therefore measured the yields of γ-radiation-induced base lesions and cluster damage, which are respectively converted into SSBs and DSBs by the base excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg). Our data demonstrate that the yield of cluster damage (i.e., lesions that yield DSBs following digestion) is 31 times higher than that of frank DSBs. This finding suggests that frank DSBs make a relatively minor contribution to the loss of DNA functionality induced by ionizing radiation, while other toxic lesions formed at a much higher frequencies than DSBs must be responsible for the loss of plasmid functionality. These lesions may be clustered lesions/locally multiply damaged sites (LMDS), including base damage, SSBs and/or intrastrand and interstrand crosslinks, leading to the loss of vital information in the DNA. Using a mathematical model, we estimate that at least three toxic lesions are required for the inactivation of plasmid functionality, in part because even these complex lesions can be repaired.

Structure and Dynamics of DNA Duplexes Containing a Cluster of Mutagenic 8-Oxoguanine and Abasic Site Lesions

Clustered DNA damage sites are caused by ionizing radiation. They are much more difficult to repair than are isolated single lesions, and their biological outcomes in terms of mutagenesis and repair inhibition are strongly dependent on the type, relative position and orientation of the lesions present in the cluster. To determine whether these effects on repair mechanism could be due to local structural properties within DNA, we used 1H NMR spectroscopy and restrained molecular dynamics simulation to elucidate the structures of three DNA duplexes containing bistranded clusters of lesions. Each DNA sequence contained an abasic site in the middle of one strand and differed by the relative position of the 8-oxoguanine, staggered on either the 3′ or the 5′ side of the complementary strand. Their repair by base excision repair protein Fpg was either complete or inhibited. All the studied damaged DNA duplexes adopt an overall B-form conformation and the damaged residues remain intrahelical. No striking deformations of the DNA chain have been observed as a result of close proximity of the lesions. These results rule out the possibility that differential recognition of clustered DNA lesions by the Fpg protein could be due to changes in the DNA's structural features induced by those lesions and provide new insight into the Fpg recognition process.

Significance of DNA polymerase I in in vivo processing of clustered DNA damage

We examined the biological consequences of bi-stranded clustered damage sites, consisting of a combination of DNA lesions, such as a 1-nucleotide gap (GAP), an apurinic/apyrimidinic (AP) site, and an 8-oxo-7,8-dihydroguanine (8-oxoG), using a bacterial plasmid-based assay. Following transformation with the plasmid containing bi-stranded clustered damage sites into the wild type strain of Escherichia coli, transformation frequencies were significantly lower for the bi-stranded clustered GAP/AP lesions (separated by 1bp) than for either a single GAP or a single AP site. When the two lesions were separated by 10–20bp, the transformation efficiencies were comparable with those of the single lesions. This recovery of transformation efficiency for separated lesions requires DNA polymerase I (Pol I) activity. Analogously, the mutation frequency was found to depend on the distance separating lesions in a bi-stranded cluster containing a GAP and an 8-oxoG, and Pol I was found to play an important role in minimising mutations induced as a result of clustered lesions. The mutagenic potential of 8-oxoG within the bi-stranded lesions does not depend on whether it is situated on the leading or lagging strand. These results indicate that the biological consequences of clustered DNA damage strongly depend on the extent of repair of the strand breaks as well as the DNA polymerase in lesion-avoidance pathways during replication.

Structural and Thermodynamic Insight into Escherichia coli UvrABC-Mediated Incision of Cluster Diacetylaminofluorene Adducts on the NarI Sequence

Cluster DNA damage refers to two or more lesions in a single turn of the DNA helix. Such clustering may occur with bulky DNA lesions, which may be responsible for their sequence-dependent repair and mutational outcomes. Here we prepared three 16-mer cluster duplexes in which two fluoroacetylaminofluorene adducts (dG-FAAF) are separated by zero, one, and two nucleotides in the Escherichia coli NarI mutational hot spot (5'-CTCTCG1G2CG3CCATCAC-3'): 5'-CG1*G2*CG3CC-3', 5'-CG1G2*CG3*CC-3', and 5'-CG1*G2CG3*CC-3' (G* = dG-FAAF), respectively. We conducted spectroscopic, thermodynamic, and molecular dynamics studies of these di-FAAF duplexes, and the results were compared with those of the corresponding mono-FAAF adducts in the same NarI sequence [Jain, V., et al. (2012) Nucleic Acids Res. 40, 3939-3951]. Our nucleotide excision repair results showed the diadducts were more reparable than the corresponding monoadducts. Moreover, we observed dramatic flanking base sequence effects on their repair efficiency in the following order: NarI-G2G3 > NarI-G1G3 > NarI-G1G2. The nuclear magnetic resonance, circular dichroism, ultraviolet melting, and molecular dynamics simulation results revealed that in contrast to the monoadducts, diadducts produced a synergistic effect on duplex destabilization. In addition, dG-FAAF at G2G3 and G1G3 destacks the neighboring bases, with greater destabilization occurring with the former. Overall, the results indicate the importance of base stacking and related thermal and thermodynamic destabilization in the repair of bulky cluster arylamine DNA adducts.

Clustered DNA damage induced by protons radiation in plasmid DNA

Clustered DNA damage is considered as a critical type of lesions induced by ionizing radiation, which can be converted into the fatal or strong mutagenic complex double strand breaks (DSBs) during damage processing in the cells. The new data show that high energy protons produce more potentially lethal DSBs than low LET radiation. In this study, plasmid DNA were used to investigate and re-evaluate the biological effects induced by the protons with the LET of ∼3.6 keV/μm at the molecular level in vitro, including single strand breaks (SSBs), DSBs, isolated and clustered base damages. The results of complex DNA damage detections indicated that protons at the given LET value induce about 1.6 fold more non-DSB clustered DNA damages than the prompt DSB. The DNA damage yields by protons were greater than that by γ-rays, specifically by 6 fold for the isolated type of DNA damage and 14 fold for the clustered damage. Furthermore, the spectrum of damages was also demonstrated to be depended on the radiation quality, with protons producing more DSBs relative to clusters than do γ-rays.

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

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

Clustered DNA damage induces pan-nuclear H2AX phosphorylation mediated by ATM and DNA–PK

DNA double-strand breaks (DSB) are considered as the most deleterious DNA lesions, and their repair is further complicated by increasing damage complexity. However, the molecular effects of clustered lesions are yet not fully understood. As the locally restricted phosphorylation of H2AX to form γH2AX is a key step in facilitating efficient DSB repair, we investigated this process after localized induction of clustered damage by ionizing radiation. We show that in addition to foci at damaged sites, H2AX is also phosphorylated in undamaged chromatin over the whole-cell nucleus in human and rodent cells, but this is not related to apoptosis. This pan-nuclear γH2AX is mediated by the kinases ataxia telangiectasia mutated and DNA-dependent protein kinase (DNA–PK) that also phosphorylate H2AX at DSBs. The pan-nuclear response is dependent on the amount of DNA damage and is transient even under conditions of impaired DSB repair. Using fluorescence recovery after photobleaching (FRAP), we found that MDC1, but not 53BP1, binds to the nuclear-wide γH2AX. Consequently, the accumulation of MDC1 at DSBs is reduced. Altogether, we show that a transient dose-dependent activation of the kinases occurring on complex DNA lesions leads to their nuclear-wide distribution and H2AX phosphorylation, yet without eliciting a full pan-nuclear DNA damage response.

Relative biological effectiveness of mammography X-rays at the level of DNA and chromosomes in lymphocytes

Purpose: In many countries, breast cancer screening programs based on periodic mammography exist, giving a large group of women regularly a small dose of ionizing radiation. In order to assess the benefit/risk ratio of those programs the relative biological effectiveness (RBE) of mammography X-rays needs to be determined.Materials and methods: Blood of five healthy donors was irradiated in vitro with 30 kV X-rays and 60Co γ-rays with doses between 5 and 2000 mGy. The phosphorylated histone subtype H2A isoform X-foci (γH2AX-foci) technique was used to quantify the number of DNA double-strand breaks (DSB) after irradiation. Chromosomal damage resulting from non- or misrepaired DNA DSB was quantified with the micronucleus (MN)-assay and the sensitivity was improved by counting only centromere negative micronuclei (MNCM−).Results: The threshold detection dose obtained with the γH2AX-foci test was 10 mGy for mammography X-rays compared to 50 mGy for γ-rays. With the MN-assay respectively MN-centromere-assay threshold detection doses of 100, respectively, 50 mGy were obtained for mammography X-rays compared to 200 respectively 100 mGy for γ-rays. An RBE of 1.4 was obtained with the γH2AX-foci assay. With the MN-assays low-dose RBE values between 3 and 4 were determined.Conclusion: Our results indicate that exposure to mammography X-rays resulted in a modest increase in the induction of DSB compared to γ-rays. However, due to the higher linear energy transfer (LET) of mammography X-rays more clustered DNA damage is produced that is more difficult to repair and results in a more pronounced increase in micronucleus formation.