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.     

Formation of strand breaks in the DNA ofγ-irradiated chromatin

Summary Strand breaks have been determined by sedimentation on sucrose gradients in the DNA of chromatin irradiated after isolation from Chinese hamster lung fibroblasts. The yields of double-strand and single-strand breaks are similar to those found in the DNA of irradiated mammalian cells. Irradiation of isolated chromatin in the presence of the radical scavenger tertiary butanol indicates that at least 65% of single-strand breaks and 56% of double-strand breaks can be attributed to the action of hydroxyl radicals. The results indicate the influence of chromosomal proteins in modifying radiation damage to DNA and suggest that the mechanisms for the induction of strand breaks in the DNA of isolated chromatin may be comparable to those operating in the intact cell.     

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     

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.     

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.     

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     

Radiation chemical mechanisms of single- and double-strand break formation in irradiated SV40 DNA

Using an electrophoresis assay system developed in our laboratory, we have simultaneously measured single- and double-strand DNA breaks (SSBs and DSBs) induced by gamma radiation in small SV40 viral DNA molecules, under conditions of greatly varying radical scavenger concentration and DNA configuration. In our experiments with aqueous solutions of SV40 DNA, we observe that SSB induction is linear with dose (one-hit response), over the entire hydroxyl scavenger efficiency range examined, from approximately 0 to 5 x 10(9) s-1, while DSB induction shifts from having a major quadratic component (two-hit response) at very low scavenger efficiencies to nearly pure linear for efficiencies greater than 10(7) s-1. The mean ratio of SSBs to one-hit DSBs remains relatively constant with increasing scavenger efficiency, decreasing from about 100:1 to 40:1 as the scavenger efficiency increases from 2 x 10(5) s-1 to 5 x 10(9) s-1, and the absolute induction efficiencies for breaks decrease by three orders of magnitude. This decrease takes place primarily at scavenger efficiencies above 1 x 10(8) s-1. Irradiation of intranuclear SV40 minichromosomes induces SSBs and DSBs at nearly the same efficiencies as does irradiation of free DNA at the highest scavenger concentrations examined, and at only about twice the efficiencies observed at -75 degrees C, where direct effects are believed to predominate. Our observations that the linear-quadratic mix of the dose-response curve for DSBs depends critically on scavenger efficiency may help to clarify the considerable confusion in the literature on the shape of such curves. Our observations of a relatively constant ratio between one-hit SSBs and DSBs at low and moderate scavenger efficiencies are in agreement with the recent hypothesis of Siddiqi and Bothe (Radiat. Res. 112, 449-463 (1987)) that, contrary to widely and long-held beliefs, the formation by indirect effects of a one-hit DSB in DNA occurs under these conditions predominantly by a mechanism involving a single OH radical, with a presumed radical transfer between complementary DNA strands. In contrast, our results for strongly protective conditions are not consistent with this hypothesis, but are consistent with the predictions of Ward's hypothesis (Radiat. Res. 86, 185-195, (1981)) that one-hit DSBs from indirect effects are produced predominantly by local clusters of OH radicals from single energy deposition events (locally multiply damaged sites) rather than by single OH radicals.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Formation of single- and double-strand breaks of pBR322 plasmid irradiated in the presence of scavengers

By the method of gel electrophoresis, radiation-induced DNA single- and double-strand breaks (SSB, DSB) were studied with a model system of pBR322 solution in vitro in the presence of ^·OH radical scavengers, mannitol and TE (10^–2 mol dm^–3 Tris and 10^–3 mol dm^–3 ethylene diamine tetra-acetic acid). Experiments showed that SSB resulted from one-hit events of radiation energy deposition and DSB resulted from both one-hit and two-hit energy deposition events and so were distinguished into two classes of αDSB and βDSB. Moreover, α/β, where α is the number of DSB per unit dose induced in one irradiation event and β the number of DSB per unit squared dose induced by the combination of two independent SSB, was related to the scavenging capacity, σ, and for σ>10⁸ s^–1,αDSB predominate over DSB. On the other hand, if σ<2×10⁸ s^–1, the measured G(αDSB) decreased in parallel with G(SSB), i.e., G(αDSB)/G(SSB) was a constant. When σ>2×10⁸ s^–1, G(αDSB) decreased slightly so that the ratio of αDSB to SSB evidently increased. Therefore, αDSB could be induced by the radical transfer mechanism for σ<2×10⁸ s^–1 and contrarily produced by the local multiply damaged sites (LMDS) mechanism for σ larger than this value. In addition, the distance for two independent complementary SSB forming a DSB was deduced, but no apparent variation of it was found in the wide σ range from ∼10⁵ to ∼10⁹ s^–1, which shows that the DNA steric structure was not influenced by mannitol. Received: 28 September 1998 / Accepted in revised form: 20 March 1999     

Formation and rejoining of deoxyribonucleic acid double-strand breaks induced in isolated cell nuclei by antineoplastic intercalating agents

The biochemical characteristics of the formation and disappearance of intercalator-induced DNA double-strand breaks (DSB) were studied in nuclei from mouse leukemia L1210 cells by using filter elution methodology [Bradley, M. O., & Kohn, K.W. (1979) Nucleic Acids Res. 7, 793-804]. The three intercalators used were 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA), 5-iminodaunorubicin (5-ID), and ellipticine. These compounds differ in that they produced predominantly DNA single-strand breaks (SSB) (m-AMSA) or predominantly DNA double-strand breaks (ellipticine) or a mixture of both SSB and DSB (5-ID) in whole cells. In isolated nuclei, each intercalator produced DSB at a frequency comparable to that which is produced in whole cells. Moreover, these DNA breaks reversed within 30 min after drug removal. It thus appeared that neither ATP nor other nucleotides were necessary for intercalator-dependent DNA nicking-closing reactions. The formation of the intercalator-induced DSB was reduced at ice temperature. Break formation was also reduced in the absence of magnesium, at a pH above 6.4 and at NaCl concentrations above 200 mM. In the presence of ATP and ATP analogues, the intercalator-induced cleavage was enhanced. These results suggest that the intercalator-induced DSB are enzymatically mediated and that the enzymes involved in these reactions can catalyze DNA double-strand cleavage and rejoining in the absence of ATP, although the occupancy of an ATP binding site might convert the enzyme to a form more reactive to intercalators. Three inhibitors of DNA topoisomerase II--novobiocin, nalidixic acid, and norfloxacin--reduced the formation of DNA strand breaks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination and analysis of site-specific 125I decay-induced DNA double-strand break end-group structures

End groups contribute to the structural complexity of radiation-induced DNA double-strand breaks (DSBs). As such, end-group structures may affect a cell's ability to repair DSBs. The 3'-end groups of strand breaks caused by gamma radiation, or oxidative processes, under oxygenated aqueous conditions have been shown to be distributed primarily between 3'-phosphoglycolate and 3'-phosphate, with 5'-phosphate ends in both cases. In this study, end groups of the high-LET-like DSBs caused by 125I decay were investigated. Site-specific DNA double-strand breaks were produced in plasmid pTC27 in the presence or absence of 2 M DMSO by 125I-labeled triplex-forming oligonucleotide targeting. End-group structure was assessed enzymatically as a function of the DSB end to serve as a substrate for ligation and various forms of end labeling. Using this approach, we have demonstrated 3'-hydroxyl (3'-OH) and 3'-phosphate (3'-P) end groups and 5'-ends (> or = 42%) terminated by phosphate. A 32P postlabeling assay failed to detect 3'-phosphoglycolate in a restriction fragment terminated by the 125I-induced DNA double-strand break, and this is likely due to restricted oxygen diffusion during irradiation as a frozen aqueous solution. Even so, end-group structure and relative distribution varied as a function of the free radical scavenging capacity of the irradiation buffer.     

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.     

Double-strand break repair in yeast requires both leading and lagging strand DNA polymerases

Mitotic double-strand break (DSB)-induced gene conversion at MAT in Saccharomyces cerevisiae was analyzed molecularly in mutant strains thermosensitive for essential replication factors. The processivity cofactors PCNA and RFC are essential even to synthesize as little as 30 nucleotides following strand invasion. Both PCNA-associated DNA polymerases delta and epsilon are important for gene conversion, though a temperature-sensitive Pol epsilon mutant is more severe than one in Pol delta. Surprisingly, mutants of lagging strand replication, DNA polymerase alpha (pol1-17), DNA primase (pri2-1), and Rad27p (rad27 delta) also greatly inhibit completion of DSB repair, even in G1-arrested cells. We propose a novel model for DSB-induced gene conversion in which a strand invasion creates a modified replication fork, involving leading and lagging strand synthesis from the donor template. Replication is terminated by capture of the second end of the DSB.     

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.     

Quantitative assessment of the contribution of clustered damage to DNA double-strand breaks induced by 60Co gamma rays and fission neutrons

The induction of DNA strand breaks by fission neutrons was studied in aqueous plasmid (pBR322) DNA under aerobic conditions for a wide range of hydroxyl radical (*OH) scavenger concentrations and was compared to the induction of strand breaks by 6OCo gamma rays. Strand breaks were measured using agarose gel electrophoresis coupled with sensitive 32P-based phosphor imaging. Yields are reported for DNA single-strand breaks (SSBs) and double-strand breaks formed linearly with dose (alphaDSBs). The fraction of alphaDSBs that were dependent on the multiply damaged site (MDS) or clustered damage mechanism was also calculated using a model. G values for SSBs and alphaDSBs declined with increasing *OH scavenging capacity. However, with increasing *OH scavenging capacities, the decrease in yields of strand breaks for fission neutrons was not as pronounced as for gamma rays. The percentage of alphaDSBs for gamma rays was dependent on *OH scavenging capacity, appearing negligible at low scavenging capacities but increasing at higher scavenging capacities. In contrast, fission neutrons induced high percentages of alphaDSBs that were approximately independent of *OH scavenging capacity. The levels of alphaDSBs formed by the MDS mechanism after exposure to fission neutrons are consistent with the expected distinctive features of high-LET energy deposition events and track structure. The results also confirm observations made by others that even for low-LET radiation, the MDS mechanism contributes significantly to DNA damage at cell-like scavenging conditions.     

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.     

Mechanistic DNA damage simulations in Geant4-DNA Part 2: Electron and proton damage in a bacterial cell

We extended a generic Geant4 application for mechanistic DNA damage simulations to an Escherichia coli cell geometry, finding electron damage yields and proton damage yields largely in line with experimental results. Depending on the simulation of radical scavenging, electrons double strand breaks (DSBs) yields range from 0.004 to 0.010 DSB Gy⁻¹ Mbp⁻¹, while protons have yields ranging from 0.004 DSB Gy⁻¹ Mbp⁻¹ at low LETs and with strict assumptions concerning scavenging, up to 0.020 DSB Gy⁻¹ Mbp⁻¹ at high LETs and when scavenging is weakest. Mechanistic DNA damage simulations can provide important limits on the extent to which physical processes can impact biology in low background experiments. We demonstrate the utility of these studies for low dose radiation biology calculating that in E. coli, the median rate at which the radiation background induces double strand breaks is 2.8 × 10⁻⁸ DSB day⁻¹, significantly less than the mutation rate per generation measured in E. coli, which is on the order of 10⁻³.     

Repair of DNA single-strand breaks in X-irradiated yeast. I. Use of the DNA-unwinding method to measure DNA strand breaks

The DNA-unwinding method developed by Ahnstr?m and his coworkers to measure DNA strand breaks in mammalian cells was used to measure single-strand breaks (SSB) in the DNA of intact yeast cells. DNA unwinding, which took place inside the rigid cell wall of yeast, was investigated as a function of time, radiation dose, and of pH and salt concentration of the alkaline solution. After DNA unwinding had taken place, the cell wall was destroyed by partial enzymatic digestion and sonication in the presence of detergents. Fragments of single- and double-stranded DNA were separated using hydroxylapatite chromatography. In this way the most suitable conditions for DNA unwinding within the cell wall were established. The results show that SSB and double-strand breaks (DSB) give rise to different kinetics of DNA unwinding.     

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.     

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.