Iron-mediated DNA damage: sensitive detection of DNA strand breakage catalyzed by iron.

Oxidative DNA damage is involved in mutagenesis, carcinogenesis, aging, radiation effects, and the action of several anticancer drugs. Accumulated evidence indicates that iron may play an important role in those processes. We studied the in vitro effect of low concentrations of Fe(II) alone or Fe(III) in the presence of reducing agents on supercoiled plasmid DNA. The assay, based on the relaxation and linearization of supercoiled DNA, is simple yet sensitive and quantitative. Iron mediated the production of single and double strand breaks in supercoiled DNA. Iron chelators, free radical scavengers, and enzymes of the oxygen reduction pathways modulated the DNA damage. Fe(III)-nitrilotriacetate (NTA) plus either H2O2, L-ascorbate, or L-cysteine produced single and double strand breaks as a function of reductant concentration. A combination of 0.1 microM Fe(III)-NTA and 100 microM L-ascorbate induced detectable DNA strand breaks after 30 min at 24 degrees C. Whereas superoxide dismutase was inhibitory only in systems containing H2O2 as reductant, catalase inhibited DNA breakage in all the iron-mediated systems studied. The effect of scavengers and enzymes indicates that H2O2 and .OH are involved in the DNA damaging process. These reactions may account for the toxicity and carcinogenicity associated with iron overload.     

Sensitivity of Deinococcus radiodurans to near-ultraviolet radiation

Although Dienococcus radiodurans is notoriously resistant to far-ultraviolet radiation (FUV; 254 nm), it is highly sensitive to near-ultraviolet radiation (NUV; 300-400 nm), thus demonstrating that the mechanisms of damage (and/or recovery) by the two types of irradiation are different. This observed difference between FUV and NUV effects in D. radiodurans agrees with previous studies with Escherichia coli. Near-ultraviolet radiation produces DNA damage which is presumed to be single-strand breaks (SSB) in the DNA of D. radiodurans. Unique lesions, such as DNA-protein crosslinks could not be demonstrated in this study. Cells that were pre-irradiated with a small dose of NUV were subsequently protected against inactivating doses of NUV. The data presented are consistent with induced DNA repair following NUV damage in D. radiodurans; this is in contrast to FUV damage where DNA repair is constitutive but not induced.     

Difference in DNA strand break by gamma- and beta-irradiations: an in vitro study

Lambda DNA (125 micrograms/ml in Tris buffer, pH 7.4) was irradiated with 60Co gamma-rays and 3H beta-rays, respectively, and the number of strand breaks was determined by electrophoresis. Number of single-strand breaks increased linearly with radiation dose in both gamma- and beta-radiations and the relative effectiveness (beta/gamma) was found to be 1.82 in N2 and 1.16 in O2. Number of double-strand breaks increased with the square of the radiation dose in gamma-irradiation, but it increased linearly with radiation dose in beta-irradiation. Therefore, the relative effectiveness (beta/gamma) is higher at lower doses. O2 effects was observed by gamma-irradiation but was minimal after beta-irradiation.     

Interaction function gamma(x) for Chinese hamster cells treated with hypertonic phosphate-buffered saline after irradiation

The repair of potentially lethal damage (PLD) in stationary-phase V79 Chinese hamster cells, which was expressible by a postirradiation treatment with hypertonic (0.5 M NaCl) phosphate-buffered saline (PBS), was analyzed within the framework of the theory of dual radiation action. The interaction function gamma(x) was estimated for cells permitted to repair PLD for various intervals of time. The experimental data indicated that 50-60% of the lethal lesions produced at the time of irradiation were repaired in 120 min. The repair of PLD was implicitly involved in the probability of the interaction of sublesions. That is, g(x,trep) was defined as the probability that two sublesions separated by distance x interact to produce a lethal lesion which will not be repaired until the fixation by treatment with hypertonic PBS at time trep after irradiation. It is concluded that the time dependence of the repair of PLD is not independent of the interaction distance x. Three conclusions are drawn: (1) The repair of a lesion produced by a long distance interaction is not detectable by postirradiation treatment with hypertonic PBS. (2) A lesion produced by a short distance interaction is rapidly repaired in about 20 min. (3) A lesion produced by the interaction of sublesions separated by a distance of about 100 nm is repaired slowly.     

Effect of radiation and freezing on [3H]DNA of Yersinia enterocolitica.

Freezing of the enteropathogenic bacterium Yersinia enterocolitica to -18 and -75 degrees C caused 7 and 42% cell death, respectively, and 0.329 and 0.588 single-strand breaks per 10(8) daltons of DNA, respectively, while radiation to one D10 dose (10% cell survival) combined with freezing to 2 to 0, -18, and -75 degrees C induced 0.05, 0.75, and 5.04 single-strand breaks, respectively. The increase in the effectiveness of radiation with respect to the yield of single-strand breaks at -18 and -75 degrees C is contrary to expectation and seems to be due to arrest of repair of single-strand breaks by these low temperatures.     

Site-specific oxidative DNA damage at polyguanosines produced by copper plus hydrogen peroxide

Oxidative DNA damage has been implicated in diverse biological processes including mutagenesis, carcinogenesis, aging, radiation effects, and chemotherapy. We examined the in vitro effect of low concentrations of Cu(II) or H2O2 alone and in combination on supercoiled plasmid DNA. As much as 10(-2) M Cu(II) or 10(-2) M H2O2 alone did not break the DNA. However, a mixture of 10(-6) M Cu(II) plus 10(-5) M H2O2 produced strand breaks and inactivated transforming ability. Strand breakage was proportional to incubation time, temperature, and Cu(II) and H2O2 concentrations. Abasic sites were not detected. Strand breakage was inhibited by metal chelators, catalase, and by high levels of free radical scavengers implying that Cu(II), Cu(I), H2O2, and .OH were involved in the reaction. The extent of DNA strand breakage was not affected by superoxide dismutase indicating that superoxide was not a major contributor to the DNA damage. DNA sequence analysis demonstrated that hot piperidine-sensitive DNA lesions were produced preferentially at sites of 2 or more adjacent guanosine residues. This sequence specificity was observed with Cu(II) plus H2O2 but not with Cu(I) alone. Polyguanosine sequence specificity for DNA damage induction appears to be unique among simple chemical systems. This reaction may be important in mechanisms of oxidative damage in vivo.     

Radioprobing of DNA: distribution of DNA breaks produced by decay of 125I incorporated into a triplex-forming oligonucleotide correlates with geometry of the triplex.

The distribution of breaks produced in both strands of a DNA duplex by the decay of 125I carried by a triplex-forming DNA oligonucleotide was studied at single nucleotide resolution. The 125I atom was located in the C5 position of a single cytosine residue of an oligonucleotide designed to form a triple helix with the target sequence duplex. The majority of the breaks (90%) are located within 10 bp around the decay site. The addition of the free radical scavenger DMSO produces an insignificant effect on the yield and distribution of the breaks. These results suggest that the majority of these breaks are produced by the direct action of radiation and are not mediated by diffusible free radicals. The frequency of breaks in the purine strand was two times higher that in the pyrimidine strand. This asymmetry in the yield of breaks correlates with the geometry of this type of triplex; the C5 of the cytosine in the third strand is closer to the sugar-phosphate backbone of the purine strand. Moreover, study of molecular models shows that the yield of breaks at individual bases correlates with distance from the 125I decay site. We suggest the possible use of 125I decay as a probe for the structure of nucleic acids and nucleoprotein complexes.     

On the chemical yield of base lesions, strand breaks, and clustered damage generated in plasmid DNA by the direct effect of X rays

The purpose of this study was to determine the yield of DNA base damages, deoxyribose damage, and clustered lesions due to the direct effects of ionizing radiation and to compare these with the yield of DNA trapped radicals measured previously in the same pUC18 plasmid. The plasmids were prepared as films hydrated in the range 2.5 < Gamma < 22.5 mol water/mol nucleotide. Single-strand breaks (SSBs) and double-strand breaks (DSBs) were detected by agarose gel electrophoresis. Specific types of base lesions were converted into SSBs and DSBs using the base-excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg). The yield of base damage detected by this method displayed a strikingly different dependence on the level of hydration (Gamma) compared with that for the yield of DNA trapped radicals; the former decreased by 3.2 times as Gamma was varied from 2.5 to 22.5 and the later increased by 2.4 times over the same range. To explain this divergence, we propose that SSB yields produced in plasmid DNA by the direct effect cannot be analyzed properly with a Poisson process that assumes an average of one strand break per plasmid and neglects the possibility of a single track producing multiple SSBs within a plasmid. The yields of DSBs, on the other hand, are consistent with changes in free radical trapping as a function of hydration. Consequently, the composition of these clusters could be quantified. Deoxyribose damage on each of the two opposing strands occurs with a yield of 3.5 +/- 0.5 nmol/J for fully hydrated pUC18, comparable to the yield of 4.1 +/- 0.9 nmol/J for DSBs derived from opposed damages in which at least one of the sites is a damaged base.     

A model of interactions between radiation-induced oxidative stress, protein and DNA damage in Deinococcus radiodurans

Ionizing radiation triggers oxidative stress, which can have a variety of subtle and profound biological effects. Here we focus on mathematical modeling of potential synergistic interactions between radiation damage to DNA and oxidative stress-induced damage to proteins involved in DNA repair/replication. When sensitive sites on these proteins are attacked by radiation-induced radicals, correct repair of dangerous DNA lesions such as double strand breaks (DSBs) can be compromised. In contrast, if oxidation of important proteins is prevented by strong antioxidant defenses, DNA repair may function more efficiently. These processes probably occur to some extent even at low doses of radiation/oxidative stress, but they are easiest to investigate at high doses, where both DNA and protein damage are extensive. As an example, we use data on survival of Deinococcus radiodurans after high doses (thousands of Gy) of acute and chronic irradiation. Our model of radiogenic oxidative stress is consistent with these data and can potentially be generalized to other organisms and lower radiation doses.     

The role of short-lived DNA lesions in the production of chromosome-exchange aberrations

Human lymphocytes were treated with combined UVC radiation and X-rays or they were X-irradiated and incubated for 60–90 min in the presence of DNA-repair inhibitor ara-C. The X-ray induced chromosome exchange aberration yield was enhanced both by UVC and ara-C by approximately a factor of two in the linear (low dose) portion of the dose-response curve. The enhancement was small in the dose squared (high dose) portion where previous dose-fractionation experiments have shown that X-ray-induced lesions leading to aberrations exist for several hours. The yield of aberrations in lymphocytes incubated after irradiation in the presence of ara-C reaches a saturation level almost immediately after irradiation (5–15 min). These cytogenetic observations together with a previous finding (Holmberg and Strausmanis, 1983) give direct and indirect evidence that the enhanced aberration yield is due to short-lived DNA breaks formed immediately after X-irradiation.

Measurements on the repair kinetics of the DNA breaks induced by X-irradiation show that ara-C strongly impairs the repair of short-lived X-ray-induced DNA breaks. It was also observed that the DNA breaks generated after UVC irradiation occur almost immediately after irradiation and the level of these transient DNA breaks reaches saturation even for short incubation times. Thus, the repair of these breaks can compete with the repair of short-lived X-ray-induced DNA-breaks in combined irradiation with UVC and X-rays.

The experimental results can be explained on the assumption that X-ray-induced aberrations originate from exchange complexes formed in interactions between both short-lived DNA breaks. The short-lived DNA breaks give rise to exchange complexes mainly within single ionization tracks where the DNA breaks are close together. The time between irradiation and exchange complex formation is of the order of 5–15 min within such a track, and short-lived breaks might be repaired before complexes have been formed. If the DNA repair of these breaks is delayed by UVC or ara-C treatment this results in a higher probability of exchange-complex formation. In contrast, interactions between breaks in different tracks originate from long-lived DNA breaks and the probability for complex formation from these breaks is not markedly affected by UVC or ara-C.     

Melanin protects melanocytes and keratinocytes against H2O2-induced DNA strand breaks through its ability to bind Ca2+

Reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)) are produced in the skin under the influence of UV radiation. These compounds are highly reactive and can induce DNA lesions in epidermal cells. Melanin is considered to protect human skin against DNA damage by absorbing UV radiation. We have investigated whether melanin can, in addition, offer protection against the effects of H(2)O(2) in human melanocytes and HaCaT keratinocytes. In the present study, it was shown that 40 and 100 microM H(2)O(2) increased the number of DNA strand breaks as measured using the comet assay, in melanocytes of Caucasian origin. In melanocytes of the same origin in which melanin levels were increased by culturing in presence of 10 mM NH(4)Cl and elevated l-tyrosine, H(2)O(2)-induced DNA damage was reduced compared to that in control melanocytes. Similarly, HaCaT cells that were loaded with melanin were better protected against H(2)O(2)-induced DNA strand breaks than control HaCaT cells. These protective effects of melanin were mimicked by the intracellular Ca(2+)-chelator BAPTA. Thus, BAPTA reduced the level of H(2)O(2)-induced DNA strand breaks in melanocytes. Like BAPTA, melanin is known to be a potent chelator of Ca(2+) and this was confirmed in the present study. It was shown that melanin levels in melanocytic cells correlated directly with intracellular Ca(2+) binding capacity and, in addition, correlated inversely with H(2)O(2)-induced increases in intracellular Ca(2+). Our results show that melanin may have an important role in regulating intracellular Ca(2+) homeostasis and it is suggested that melanin protects against H(2)O(2)-induced DNA strand breaks in both melanocytes and keratinocytes and through its ability to bind Ca(2+).     

A generalized formulation of dual radiation action

Dual radiation action is a process in which cellular lesions are produced as a result of the interaction of pairs of sublesions that are molecular alterations produced by ionizing radiation. Previous formulations of this process have employed a number of simplifying assumptions that limit the accuracy and the range of application of theoretical analysis. The formulation presented here removes some of these restrictions by introducing three functions that describe the geometry of the sensitive material in the cell, the geometry of the pattern of energy deposition, and the interaction probability of sublesions as a function of their separation. The relation derived is similar to that obtained previously, in that lesion production is found to depend on two terms that are proportional to the first and the second power of the absorbed dose. However, the coefficients of these terms are now derived on the basis of a more realistic treatment.     

激光对DNA作用机理的AFM研究

激光作用质粒DNA和小牛胸腺DNA产生损伤效应,导致DNA结构变化,利用一种改进的试样制备过程和纳米显微镜－－原子力显微镜（AFM）能够获得可重现的激光作用质粒DNA和小牛胸腺DNA的AFM图像,显示它们的特殊的表达结构,讨论了激光辐照导致DNA链断裂的作用机理。     

Effect of 3-aminobenzamide on DNA strand-break rejoining and cytotoxicity in CHO cells treated with hydrogen peroxide

The influence of the nuclear ADP-ribosyltransferase inhibitor 3-aminobenzamide on the DNA strand-break rejoining kinetics and cytotoxicity in Chinese hamster ovary cells following H2O2 treatment was investigated. For the DNA damage studies, cells were treated on ice with H2O2 (0-20 microM) for 1 h in serum-free medium, after which the H2O2 was removed and the cells were allowed to repair their damage in complete medium at 37 degrees C in the presence or absence of 3-aminobenzamide (5 mM) for periods up to 2 h. The DNA strand breaks remaining as a function of time were then estimated by alkaline elution. A linear relationship between the H2O2 concentration and the initial level of DNA single-strand breaks (zero time allowed for repair) was observed. No double-strand breaks or DNA-protein cross-links were detected at these doses. The rejoining of single-strand breaks after H2O2 (20 microM) alone was characterized by a single exponential process with a t1/2 of approx. 5 min. However, in the presence of 3-aminobenzamide, rejoining was much slower and biphasic, with t1/2 of approx. 10 and 36 min. The inhibitory action of 3-aminobenzamide was concentration-dependent and completely reversible in that, when the 3-aminobenzamide was removed from the treated cultures, the strand-break rejoining kinetics rapidly returned to the t1/2 of 5 min typical of H2O2 alone. Considerably higher concentrations of H2O2 (up to 600 microM) were required for cell killing compared to the DNA damage studies. Cell killing by H2O2 alone was characterized by a shoulderless, exponential survival curve (D0 = 880 microM). The cytotoxicity was potentiated when the cells were treated with 3-aminobenzamide (5 mM) for 1 h after the H2O2 treatment; the survival curve with 3-aminobenzamide also assumed a biphasic character (D0 of 212 microM and 520 microM). These results are consistent with the theory that OH.-induced single-strand breaks do not normally represent lethal lesions to the cell because of their rapid, efficient repair. However, interference with these repair processes (in this case by 3-aminobenzamide) can alter this relationship, possibly allowing lesion fixation.     

Studies on mutagen-sensitive strains of Drosophila melanogaster. X. Repair of radiation-induced DNA damage in primary cell cultures after irradiation with X-rays

The repair of X-ray-induced DNA lesions in repair-deficient mutant strains was studied as a way of investigating the mechanism of the induction of genetic damage. Genetic effects on the recovery of X-ray-induced damage by the repair-deficient strains ebony (photoreactivation repair-deficient) and mus(1)101D1 (post-replication repair-deficient) were interpreted as impaired repair of single- and double-strand DNA breaks. We investigated the repair of X-ray-induced DNA breaks and alkaline-labile sites in primary cell cultures of ebony and mus(1)101D1 and in cultures of their control strains. No significant differences were found between the repair rates in the mutants and control strains. This indicates that the genetic effects of these mutants are not due to an impaired rate of repair of DNA breaks.     

X-ray-induced base sequence damage in primate alphoid DNA

Radiation-induced single-strand breaks were found throughout the 172 bp repeat units of African green monkey component alpha DNA. Two kinds of 3'-ends of 5'-32P-labeled restriction fragments were found, as previously described by others. After irradiation in vitro, the yield of single-strand breaks was 4 X 10(-5) breaks/nucleotide/Gy, as determined by analyses in DNA sequencing type gels. Protection from X-ray damage was found when the DNA received 150 Gy in the presence of 2-mercaptoethanol. The results demonstrate a very sensitive quantitative means to study the role of indirect effects of ionizing radiation on strand-break induction and protection at the base sequence level. Component alpha DNA was isolated from irradiated CV-1 cells and was analyzed for single-strand breaks. Under these conditions the frequency of breaks was less than the frequency obtained when purified DNA was irradiated. The methodology is presented because of its relevance to the study of DNA strand breakage in living cells.     

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.     

NEIL1 is the major DNA glycosylase that processes 5-hydroxyuracil in the proximity of a DNA single-strand break

5-Hydroxyuracil (5-OHU) in DNA, arising during endogenous DNA damage and caused by ionizing radiation, is removed by the base excision repair pathway. However, in addition to base lesions, ionizing radiation also generates DNA single-strand breaks (SSBs). When these DNA lesions are located in the proximity of each other, this may result in a profound effect on both repair of the damaged base and the SSB. We therefore examined the repair of DNA substrates containing 5-OHU lesions in the proximity of the 3'-end of a SSB. We found that SSB repair by DNA ligase IIIalpha and DNA polymerase beta is impaired by the presence of the nearby 5-OHU lesion, indicating the requirement for a DNA glycosylase which would be able to remove 5-OHU before SSB repair. Subsequently, we found that although both SMUG1 and NEIL1 are able to excise 5-OHU lesions located in the proximity of the 3'-end of a DNA SSB, NEIL1 is more efficient in the repair of these DNA lesions.     

Strange goings-on in the mouse germ line

It is a conventional paradigm that mutagens lead to changes in nucleotide sequence when the cell attempts to repair or replicate lesions in DNA (such as adducts or strand breaks) that have been produced by the mutagens or their metabolites. The resulting changes are located at (or very near) the sites of the initial damage. This is the underlying theory behind mutational spectra work, but how general is it in vivo? Work with ionising radiation has shown that there are interesting things going on in the mouse germ line that do not fall within the conventional paradigm. Mutations occur at certain sites remote from initial DNA damage and in greater than expected number. Bryn Bridges discusses some recent papers on mutational changes in the germ line of mice following exposure to chemical mutagens that suggest that such phenomena may not be confined to radiation.     

Involvement of DNA polymerase beta in repair of ionizing radiation damage as measured by in vitro plasmid assays

Characteristic of damage introduced in DNA by ionizing radiation is the induction of a wide range of lesions. Single-strand breaks (SSBs) and base damages outnumber double-strand breaks (DSBs). If unrepaired, these lesions can lead to DSBs and increased mutagenesis. XRCC1 and DNA polymerase beta (polbeta) are thought to be critical elements in the repair of these SSBs and base damages. XRCC1-deficient cells display a radiosensitive phenotype, while proliferating polbeta-deficient cells are not more radiosensitive. We have recently shown that cells deficient in polbeta display increased radiosensitivity when confluent. In addition, cells expressing a dominant negative to polbeta have been found to be radiosensitized. Here we show that repair of radiation-induced lesions is inhibited in extracts with altered polbeta or XRCC1 status, as measured by an in vitro repair assay employing irradiated plasmid DNA. Extracts from XRCC1-deficient cells showed a dramatically reduced capacity to repair ionizing radiation-induced DNA damage. Extracts deficient in polbeta or containing a dominant negative to polbeta also showed reduced repair of radiation-induced SSBs. Irradiated repaired plasmid DNA showed increased incorporation of radioactive nucleotides, indicating use of an alternative long-patch repair pathway. These data show a deficiency in repair of ionizing radiation damage in extracts from cells deficient or altered in polbeta activity, implying that increased radiosensitivity resulted from radiation damage repair deficiencies.