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.     

Further evidence for double-strand breaks originating from a paired radical precursor from studies of oxygen fixation processes.

By using a fast reaction technique which employs H2S gas as a fast-reacting chemical repair agent, it is possible to measure the competition kinetics between chemical repair reactions and oxygen fixation reactions in model DNA and cellular systems. In plasmid pBR322 DNA irradiated with electrons, we have compared the oxygen fixation reactions of the free radical precursors that lead to the production of single-strand (SSBs) and double-strand breaks (DSBs). For the oxygen-dependent fixation of radical damage leading to SSBs, a second-order rate constant of 2.3 x 10(8) dm3 mol(-1) s(-1) was obtained compared to 8.9 x 10(7) dm3 mol(-1) s(-1) for DSBs. The difference is in general agreement with predictions from a multiple-radical model where the precursor of a DSB originates from two radicals. The fixation of this precursor by oxygen will require both radicals to be fixed for the DSB to be formed, which will have slower kinetics than that of single free-radical precursors of SSBs.     

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.     

Protection of DNA from high LET radiation by two OH radical scavengers,tris (hydroxymethyl) aminomethane and 2-mercaptoethanol

The effect of high linear energy transfer (LET) radiation on DNAin vitro, both in protective and non-protective environments was investigated. Two hydroxyl radical scavengers, tris(hydroxymethyl) aminomethane and 2-mercaptoethanol, were compared for their ability to protect SV40 DNA from radiation damage over a wide LET range. At comparable OH scavenging capacities, significant differences were found between these protective agents, indicating that other, radical scavenger-dependent processes affected the extent to which the DNA was protected. In general, a decrease in single-strand breaks (SSBs) relative to double-strand breaks (DSBs) was observed as LET increased. This effect was more pronounced when a radioprotector was present. Comparison of the relative biological efficiency (RBE) of radiation damage as LET increased showed a peak of DSB production in the mid-LET range. These data agree with measurements made by Christensen et al. (1972). An explanation for this increase in DSB production efficiency has been proposed based on the particle track structure of high-LET radiation.Correspondence to: G. Taucher-Scholz     

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.     

Direct effect in DNA radiolysis. Boron neutron capture enhancement of radiolysis in a medical fast-neutron beam

SècheThis paper is devoted to the study of the molecular basis of the boron neutron capture enhancement of fast-neutron radiotherapy. Plasmid DNA was irradiated with a medical fast-neutron beam in the presence of either (10)B or (11)B. The number of induced SSBs and DSBs was much higher in samples containing (10)B compared to (11)B. The additional breaks are attributed to the nuclear reaction (10)B(n, alpha)(7)Li induced by the capture by (10)B of thermal neutrons produced in the medium by scattering and slowing down of neutrons. Irradiation in the presence of DMSO (OH radical scavenger) allows the number of nonscavengeable breaks to be determined. The ratio DSB/SSB is within the range of those observed with heavy ions, in good agreement with the hypothesis that the additional breaks are due to alpha particles and recoil lithium nuclei. The simulation of the energy deposition along the paths of the alpha and (7)Li particles allows the calculation of core and penumbra track volumes. Further, the number of plasmids encountered by the core and the penumbra was evaluated. Their number was compared to the nonscavengeable additional breaks. Since the two sets of values are of the same order of magnitude, we conclude that the nonscavengeable additional SSBs and DSBs could be due to direct effects.     

Quantification of dye-mediated photodamage during single-molecule DNA imaging

Single-molecule fluorescence imaging of DNA-binding proteins has enabled detailed investigations of their interactions. However, the intercalating dyes used to visually locate DNA molecules have the undesirable effect of photochemically damaging the DNA through radical intermediaries. Unfortunately, this damage occurs as single-strand breaks (SSBs), which are visually undetectable but can heavily influence protein behavior. We investigated the formation of SSBs on DNA molecules by the dye YOYO-1 using complementary single-molecule imaging and gel electrophoresis-based damage assays. The single-molecule assay imaged hydrodynamically elongated lambda DNA, enabling the real-time detection of double-strand breaks (DSBs). The gel assay, which used supercoiled plasmid DNA, was sensitive to both SSBs and DSBs. This enabled the quantification of SSBs that precede DSB formation. Using the parameters determined from the gel damage assay, we applied a model of stochastic DNA damage to the time-resolved DNA breakage data, extracting the rates of single-strand breakage at two dye staining ratios and measuring the damage reduction from the radical scavengers ascorbic acid and β-mercaptoethanol. These results enable the estimation of the number of SSBs that occur during imaging and are scalable over a wide range of laser intensities used in fluorescence microscopy.     

Repair of radiation-induced heat-labile sites is independent of DNA-PKcs, XRCC1 and PARP

Ionizing radiation induces a variety of different DNA lesions; in addition to the most critical DNA damage, the DSB, numerous base alterations, SSBs and other modifications of the DNA double-helix are formed. When several non-DSB lesions are clustered within a short distance along DNA, or close to a DSB, they may interfere with the repair of DSBs and affect the measurement of DSB induction and repair. We have shown previously that a substantial fraction of DSBs measured by pulsed-field gel electrophoresis (PFGE) are in fact due to heat-labile sites within clustered lesions, thus reflecting an artifact of preparation of genomic DNA at elevated temperature. To further characterize the influence of heat-labile sites on DSB induction and repair, cells of four human cell lines (GM5758, GM7166, M059K, U-1810) with apparently normal DSB rejoining were tested for biphasic rejoining after gamma irradiation. When heat-released DSBs were excluded from the measurements, the fraction of fast rejoining decreased to less than 50% of the total. However, the half-times of the fast (t(1/2) = 7-8 min) and slow (t(1/2) = 2.5 h) DSB rejoining were not changed significantly. At t = 0, the heat-released DSBs accounted for almost 40% of the DSBs, corresponding to 10 extra DSBs per cell per Gy in the initial DSB yield. These heat-released DSBs were repaired within 60-90 min in all cells tested, including M059K cells treated with wortmannin and DNA-PKcs-defective M059J cells. Furthermore, cells lacking XRCC1 or poly(ADP-ribose) polymerase 1 (PARP1) rejoined both total DSBs and heat-released DSBs similarly to normal cells. In summary, the presence of heat-labile sites has a substantial impact on DSB induction and DSB rejoining rates measured by pulsed-field gel electrophoresis, and heat-labile sites repair is independent of DNA-PKcs, XRCC1 and PARP.     

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.     

Single- and double-strand break formation in DNA irradiated in aqueous solution: dependence on dose and OH radical scavenger concentration

The yields of single- and double-strand breaks (SSB and DSB) in calf thymus DNA, after 60Co gamma irradiation in dilute aqueous solution, have been determined via molecular weight measurements using a low-angle laser light scattering technique. The irradiations were administered to N2O-containing solutions of DNA in the absence and presence of oxygen and with different concentrations of the OH radical scavengers phenol, tertiary butanol, and methanol. OH radicals were found to produce SSB linearly with dose with a G value of 55 nmol J-1 and 54 nmol J-1 in deoxygenated and oxygenated solutions, respectively. DSB were formed according to a linear-quadratic dose relationship and the G value of linearly formed DSB were GDSB alpha(r.t.) = 3.5 nmol J-1 in deoxygenated and 3.2 nmol J-1 in oxygenated solution. The ratio of GSSB/GDSB alpha(r.t.) = gamma of 19 +/- 6 was independent of the scavenger concentration in the case of tertiary butanol and methanol-containing solutions. GDSB alpha(r.t.) is interpreted to result from a radical site transferred from a sugar moiety of the cleaved strand to the complementary intact strand. This process of radical transfer and subsequent cleavage of the second strand occurs with a probability of about 6 +/- 2% in the presence of oxygen at all scavenger concentrations studied. These data on scavenging capacity on GDSB alpha(r.t.) suggest that the double-strand breakage produced via radical transfer remains higher than that resulting from direct effect, up to scavenging capacities of about 10(9) s-1.     

Elaboration of cellular DNA breaks by hydroperoxides.

Cellular damage produced by ionizing radiation and peroxides, hydrogen peroxide (HOOH) and the organic peroxides tert-butyl (tBuOOH) or cumene hydroperoxide (CuOOH) were compared. DNA breaks, toxicity, malondialdehyde production, and the rate of peroxide disappearance were measured in a human adenocarcinoma cell line (A549). The alkaline and neutral filter elution assays were used to quantitate the kinetics of single and double strand break formation and repair (SSB and DSB), respectively. Peroxides, at 0.01-1.0 mM, produce multiphasic dose response curves for both toxicity and DNA SSBs. Radiation, 1-6 Gy, produced a shouldered survival curve, and both DNA SSB and DSBs produced in cells x-rayed on ice were nearly linear with dose. The peroxides produced more SSBs than radiation at equitoxic doses. X-ray induced DNA single strand breaks were rejoined rapidly by cells at 37 degrees C with approximately 80% of initial damage repaired in 20 min. Peroxide induced SSBs were maximal after 15 min at 37 degrees C. Rejoining proceeded thereafter, but at a rate less than for x-ray induced strand breaks. Significant DNA DSBs could not be achieved by peroxides even at concentrations 50-fold higher than required to produce SSBs. HOOH treatment of DNA on filters following cell lysis and proteolysis produced SSBs. CuOOH and tBuOOH produced no SSBs in lysed cell DNA. None of the peroxides produced DSBs when incubated with lysed cell DNA. Malondialdehyde was released from cells incubated with organic hydroperoxides, but not HOOH, nor up to 40 Gy of x-rays. HOOH was metabolized three times faster than the organic peroxides. The overall results demonstrate the necessity for a metabolically active cell environment to elaborate maximal DNA strand breaks and cell death at hydroperoxide concentrations of 10(-4) or greater, but prevent strand breaks and stimulate cell growth at 10(-5) M.     

Radiation induced DNA DSBs: Contribution from stalled replication forks?

When cells are exposed to radiation serious lesions are introduced into the DNA including double strand breaks (DSBs), single strand breaks (SSBs), base modifications and clustered damage sites (a specific feature of ionizing radiation induced DNA damage). Radiation induced DNA damage has the potential to initiate events that can lead ultimately to mutations and the onset of cancer and therefore understanding the cellular responses to DNA lesions is of particular importance. Using γH2AX as a marker for DSB formation and RAD51 as a marker of homologous recombination (HR) which is recruited in the processing of frank DSBs or DSBs arising from stalled replication forks, we have investigated the contribution of SSBs and non-DSB DNA damage to the induction of DSBs in mammalian cells by ionizing radiation during the cell cycle. V79-4 cells and human HF19 fibroblast cells have been either irradiated with 0–20 Gy of γ radiation or, for comparison, treated with a low concentration of hydrogen peroxide, which is known to induce SSBs but not DSBs. Inhibition of the repair of oxidative DNA lesions by poly(ADP ribose) polymerase (PARP) inhibitor leads to an increase in radiation induced γH2AX and RAD51 foci which we propose is due to these lesions colliding with replication forks forming replication induced DSBs. It was confirmed that DSBs are not induced in G₁ phase cells by treatment with hydrogen peroxide but treatment does lead to DSB induction, specifically in S phase cells. We therefore suggest that radiation induced SSBs and non-DSB DNA damage contribute to the formation of replication induced DSBs, detected as RAD51 foci.     

Age and gender effects on DNA strand break repair in peripheral blood mononuclear cells

Exogenous and endogenous damage to DNA is constantly challenging the stability of our genome. This DNA damage increase the frequency of errors in DNA replication, thus causing point mutations or chromosomal rearrangements and has been implicated in aging, cancer, and neurodegenerative diseases. Therefore, efficient DNA repair is vital for the maintenance of genome stability. The general notion has been that DNA repair capacity decreases with age although there are conflicting results. Here, we focused on potential age‐associated changes in DNA damage response and the capacities of repairing DNA single‐strand breaks (SSBs) and double‐strand breaks (DSBs) in human peripheral blood mononuclear cells (PBMCs). Of these lesions, DSBs are the least frequent but the most dangerous for cells. We have measured the level of endogenous SSBs, SSB repair capacity, γ‐H2AX response, and DSB repair capacity in a study population consisting of 216 individuals from a population‐based sample of twins aged 40–77 years. Age in this range did not seem to have any effect on the SSB parameters. However, γ‐H2AX response and DSB repair capacity decreased with increasing age, although the associations did not reach statistical significance after adjustment for batch effect across multiple experiments. No gender differences were observed for any of the parameters analyzed. Our findings suggest that in PBMCs, the repair of SSBs is maintained until old age, whereas the response to and the repair of DSBs decrease.     

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.     

Activation of ataxia telangiectasia mutated by DNA strand break-inducing agents correlates closely with the number of DNA double strand breaks

The protein kinase ataxia telangiectasia mutated (ATM) is activated when cells are exposed to ionizing radiation (IR). It has been assumed that ATM is specifically activated by the few induced DNA double strand breaks (DSBs), although little direct evidence for this assumption has been presented. DSBs constitute only a few percent of the IR-induced DNA damage, whereas the more frequent single strand DNA breaks (SSBs) and base damage account for over 98% of the overall DNA damage. It is therefore unclear whether DSBs are the only IR-induced DNA lesions that activate ATM. To test directly whether or not DSBs are responsible for ATM activation, we exposed cells to drugs and radiation that produce different numbers of DSBs and SSBs. We determined the resulting ATM activation by measuring the amount of phosphorylated Chk2 and the numbers of SSBs and DSBs in the same cells after short incubation periods. We found a strong correlation between the number of DSBs and ATM activation but no correlation with the number of SSBs. In fact, hydrogen peroxide, which, similar to IR, induces DNA damage through hydroxyl radicals but fails to induce DSBs, did not activate ATM. In contrast, we found that calicheamicin-induced strand breaks activated ATM more efficiently than IR and that ATM activation correlated with the relative DSB induction by these agents. Our data indicate that ATM is specifically activated by IR-induced DSBs, with little or no contribution from SSBs and other types of DNA damage. These findings have implications for how ATM might recognize DSBs in cells.     

Effects of DNA double-strand and single-strand breaks on intrachromosomal recombination events in cell-cycle-arrested yeast cells.

Intrachromosomal recombination between repeated elements can result in deletion (DEL recombination) events. We investigated the inducibility of such intrachromosomal recombination events at different stages of the cell cycle and the nature of the primary DNA lesions capable of initiating these events. Two genetic systems were constructed in Saccharomyces cerevisiae that select for DEL recombination events between duplicated alleles of CDC28 and TUB2. We determined effects of double-strand breaks (DSBs) and single-strand breaks (SSBs) between the duplicated alleles on DEL recombination when induced in dividing cells or cells arrested in G1 or G2. Site-specific DSBs and SSBs were produced by overexpression of the I-Sce I endonuclease and the gene II protein (gIIp), respectively. I-Sce I-induced DSBs caused an increase in DEL recombination frequencies in both dividing and cell-cycle-arrested cells, indicating that G1- and G2-arrested cells are capable of completing DSB repair. In contrast, gIIp-induced SSBs caused an increase in DEL recombination frequency only in dividing cells. To further examine these phenomena we used both gamma-irradiation, inducing DSBs as its most relevant lesion, and UV, inducing other forms of DNA damage. UV irradiation did not increase DEL recombination frequencies in G1 or G2, whereas gamma-rays increased DEL recombination frequencies in both phases. Both forms of radiation, however, induced DEL recombination in dividing cells. The results suggest that DSBs but not SSBs induce DEL recombination, probably via the single-strand annealing pathway. Further, DSBs in dividing cells may result from the replication of a UV or SSB-damaged template. Alternatively, UV induced events may occur by replication slippage after DNA polymerase pausing in front of the damage.     

99mTc-Labeled HYNIC-DAPI Causes Plasmid DNA Damage with High Efficiency

^99mTc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, ^99mTc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs) or double-strand breaks (DSBs); the resulting DNA fragments can be separated by gel electrophoresis and quantified by fluorescent staining. This study aimed to compare the plasmid DNA damage potential of a ^99mTc-labeled HYNIC-DAPI compound with that of ^99mTc pertechnetate (^99mTcO₄ ⁻). pUC19 plasmid DNA was irradiated for 2 or 24 hours. Direct and radical-induced DNA damage were evaluated in the presence or absence of the radical scavenger DMSO. For both compounds, an increase in applied activity enhanced plasmid DNA damage, which was evidenced by an increase in the open circular and linear DNA fractions and a reduction in the supercoiled DNA fraction. The number of SSBs elicited by ^99mTc-HYNIC-DAPI (1.03) was twice that caused by ^99mTcO₄ ⁻ (0.51), and the number of DSBs increased fivefold in the ^99mTc-HYNIC-DAPI-treated sample compared with the ^99mTcO₄ ⁻ treated sample (0.02 to 0.10). In the presence of DMSO, the numbers of SSBs and DSBs decreased to 0.03 and 0.00, respectively, in the ^99mTcO₄ ^– treated samples, whereas the numbers of SSBs and DSBs were slightly reduced to 0.95 and 0.06, respectively, in the ^99mTc-HYNIC-DAPI-treated samples. These results indicated that ^99mTc-HYNIC-DAPI induced SSBs and DSBs via a direct interaction of the ^99mTc-labeled compound with DNA. In contrast to these results, ^99mTcO₄ ⁻ induced SSBs via radical formation, and DSBs were formed by two nearby SSBs. The biological effectiveness of ^99mTc-HYNIC-DAPI increased by approximately 4-fold in terms of inducing SSBs and by approximately 10-fold in terms of inducing DSBs.     

Variation in radiation-induced formation of DNA double-strand breaks as a function of chromatin structure.

The influence of chromatin structure on induction of DNA double-strand breaks (DSBs) by X radiation was studied in DNA from CHO cells. Whole cells, nuclei with condensed or relaxed chromatin, and deproteinized DNA in agarose plugs were irradiated and DSB formation was measured as a decrease in the length of DNA by nondenaturing, pulsed-field, agarose gel electrophoresis. The yield of DSBs in deproteinized DNA (2.3 x 10(-10) DSBs Da-1 Gy-1) was observed to be 70 times greater than the yield of DSBs (3.1 x 10(-12) DSBs Da-1 Gy-1) observed in DNA in the intact cell nucleus. Organization of DNA into the basic nucleosome repeat structure and condensation of the chromatin fiber into higher-order structure protected DNA from DSB induction by factors of 8.3 and 4.5, respectively. An additional twofold protection of DNA in fully condensed chromatin occurred in the intact cell nucleus. Since this protection did not appear to involve chromatin structure, we speculate that this additional protection may result from the association of soluble protein and nonprotein sulfhydryls with DNA in the intact cell nucleus. The results are consistent with the organization of nuclear DNA into both basic nucleosome repeat structure and higher-order chromatin structure providing significant protection against DSB induction.     

Unwinding of origin-specific structures by human replication protein A occurs in a two-step process.

The simian virus 40 (SV40) large tumor antigen(T antigen) has been shown to induce the melting of 8 bp within the SV40 origin of replication. We found previously that a 'pseudo-origin' DNA molecule (PO-8) containing a central 8 nt single-stranded DNA (ssDNA) bubble was efficiently bound and denatured by human replication protein A (hRPA). To understand the mechanism by which hRPA denatures these pseudo-origin molecules, as well as the role that hRPA plays during the initiation of SV40 DNA replication, we characterized the key parameters for the pseudo-origin binding and denaturation reactions. The dissociation constant of hRPA binding to PO-8 was observed to be 7.7 x 10(-7) M, compared to 9.0 x 10(-8) M for binding to an identical length ssDNA under the same reaction conditions. The binding and denaturation of PO-8 occurred with different kinetics with the rate of binding determined to be approximately 4-fold greater than the rate of denaturation. Although hRPA binding to PO-8 was relatively temperature independent, an increase in incubation temperature from 4 to 37 degreesC stimulated denaturation nearly 4-fold. At 37 degreesC, denaturation occurred on approximately 1/3 of those substrate molecules bound by hRPA, showing that hRPA can bind the pseudo-origin substrate without causing its complete denaturation. Tests of other single-stranded DNA-binding proteins (SSBs) over a range of SSB concentrations revealed that the ability of the SSBs to bind the pseudo-origin substrate, rather than denature the substrate, correlated best with the known ability of these SSBs to support the T antigen-dependent SV40 origin-unwinding activity. Our data indicate that hRPA first binds the DNA substrate using a combination of contacts with the ssDNA bubble and duplex DNA flanks and then, on only a fraction of the bound substrate molecules, denatures the DNA substrate.     

Cell inactivation and double-strand breaks: the role of core ionizations, as probed by ultrasoft X rays

The large RBE (approximately 7) measured for the killing of Chinese hamster V79 cells by 340 eV ultrasoft X rays, which preferentially ionize the K shell of carbon atoms (Hervé du Penhoat et al., Radiat. Res. 151, 649-658, 1999), was used to investigate the location of sensitive sites for cell inactivation and the physical modes of action of radiation. The enhancement of the RBE above the carbon K-shell edge either may indicate a high intrinsic efficiency of carbon K-shell ionizations (due, for example, to a specific physical or chemical effect) or may be related to the preferential localization of these ionizations on the DNA. The second interpretation would indicate a strong local (within 3 nm) action of K-shell ionizations and consequently the importance of a direct mechanism for radiation lethality (without excluding an action in conjunction with an indirect component). To distinguish between these two hypotheses, the efficiencies of core ionizations in DNA atoms (phosphorus L-shell, carbon K-shell, and oxygen K-shell ionizations) to induce damages were investigated by measuring their capacities to produce DNA double-strand breaks (DSBs). The effect of photoionizations in isolated DNA was studied using pBS plasmids in a partially hydrated state. No enhancement of the efficiency of DSB induction by carbon K-shell ionizations compared to oxygen K-shell ionizations was found, supporting the hypothesis that it is the localization of these carbon K-shell events on DNA which gives to the 340 eV photons their high killing efficiency. In agreement with this interpretation, cell inactivation and DSB induction, which do not appear to be correlated when expressed in terms of yields per unit dose in the sample, exhibit a rather good correlation when expressed in terms of efficiencies per core event in the DNA. These results suggest that core ionizations in DNA, through core-hole relaxation in conjunction with localized effects of spatially correlated secondary and Auger electrons, may be the major critical events for cell inactivation, and that the resulting DSBs (or a constant fraction of these DSBs) may be a major class of unrepairable lesions.