Ayumi Urushibara

LET dependence of the yield of single-, double-strand breaks and base lesions in fully hydrated plasmid DNA films by 4He2+ ion irradiation

Purpose: To characterize the complexity of DNA damage through determination of the yields of single (SSB) and double strand breaks (DSB), base lesions and clustered damage sites induced in fully hydrated plasmid DNA by direct radiation effects as a function of the ionizing density of the radiation using 4He2+ ion irradiation with linear energy-transfer (LET) values in the range 19 to 148 keV/μm. Materials and methods: Hydrated plasmid DNA (pUC18) containing 34.5 water molecules/nucleotide was irradiated with He2+ ions with LET values of 19, 63, 95, 121 and 148 keV/μm. From quantification of the conformational changes of the irradiated samples (closed circular, open or linear forms) analyzed by agarose gel electrophoresis, the yields of SSB and DSB were obtained. Base lesions were visualized as additional strand breaks by treatment with base excision repair enzymes (endonuclease III (Nth) and formamidpyrimidine DNA glycosylase (Fpg)). Results: The yield of prompt SSB does not depend significantly on LET of the 4He2+ ions, whereas the yield of prompt DSB increases with increasing LET. The yields of isolated base lesions, revealed by Nth and Fpg as additional SSB, decrease drastically with increasing LET. The sum of the yields of DSB and additional DSB revealed by Nth and Fpg increase with increasing LET of the 4He2+ ions except at the highest LET investigated. Conclusion: The yields of clustered damage, revealed as DSB and non-DSB clustered damage sites, but not isolated lesions, namely SSB, increase with increasing ionization density of the 4He2+ ions except at the highest LET investigated.

The mutagenic potential of 8-oxoG/single strand break-containing clusters depends on their relative positions.

The biological consequences of clusters containing a single strand break and base lesion(s) remain largely unknown. In the present study we determined the mutagenicities of two- and three-lesion clustered damage sites containing a 1-nucleotide gap (GAP) and 8-oxo-7,8-dihydroguanine(s) (8-oxoG(s)) in Escherichia coli. The mutation frequencies (MFs) of bi-stranded two-lesion clusters (GAP/8-oxoG), especially in mutY-deficient strains, were high and were similar to those for bi-stranded clusters with 8-oxoG and base lesions/AP sites, suggesting that the GAP is processed with an efficiency similar to the efficiency of processing a base lesion or an AP site within a cluster. The MFs of tandem two-lesion clusters comprised of a GAP and an 8-oxoG on the same strand were comparable to or less than the MF of a single 8-oxoG. The mutagenic potential of three-lesion clusters, which were comprised of a tandem lesion (a GAP and an 8-oxoG) and an opposing single 8-oxoG, was higher than that of a single 8-oxoG, but was no more than that of a bi-stranded 8-oxoGs. We suggest that incorporation of a nucleotide opposite 8-oxoG is less mutagenic when a GAP is present in a cluster than when a GAP is absent. Our observations indicate that the repair of a GAP is retarded by an opposing 8-oxoG, but not by a tandem 8-oxoG, and that the extent of GAP repair determines the biological consequences.

Studies of soft X-ray-induced Auger effect on the induction of DNA damage.

Purpose: To understand the characteristics of DNA damage induced by Auger effect in DNA by ultrasoft X-irradiation. In situ electron paramagnetic resonance (EPR) spectroscopy as well as biochemical analysis has been applied to examine the DNA damage induction in both viewpoints of intermediate species and final products. Materials and methods: Unpaired electron species induced in a calf thymus DNA film irradiated with monochromatic ultrasoft X-rays (270–580 eV) was observed using an X-band EPR spectrometer installed in a synchrotron beamline. To determine the yield of single strand break (SSB), pUC18 plasmid DNA was irradiated and then analyzed by agarose gel electrophoresis. To analyze molecular change in a single strand DNA, a new technique using DNA-denaturation-treatment has been applied to quantify multiple SSB arising in both DNA strands. Results: Short-lived EPR spectra were observed during irradiation. The intensity of transient EPR spectrum shows the similar energy dependence with that of the SSB yield around oxygen K-edge in particular. The fraction of the single-strand plasmid DNA (SS-DNA) after irradiation could be determined using a low-temperature–denaturation condition. The obtained slope of the dose-response for SS-DNA shows half of that of closed circular DNA as expected under the diluted solution condition. Conclusion: The availability of an EPR apparatus installed in a synchrotron beamline is demonstrated by detecting very short-lived unpaired electron species. Transient EPR spectra of DNA show the similar energy dependence to that of the SSB yield. The proposed DNA-denaturation assay works as expected using the low-temperature–denaturation condition.

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.

Chemical repair of base lesions, AP-sites, and strand breaks on plasmid DNA in dilute aqueous solution by ascorbic acid

We quantified the damage yields produced in plasmid DNA by γ-irradiation in the presence of low concentrations (10-100 μM) of ascorbic acid, which is a major antioxidant in living systems, to clarify whether it chemically repairs radiation damage in DNA. The yield of DNA single strand breaks induced by irradiation was analyzed with agarose gel electrophoresis as conformational changes in closed circular plasmids. Base lesions and abasic sites were also observed as additional conformational changes by treating irradiated samples with glycosylase proteins. By comparing the suppression efficiencies to the induction of each DNA lesion, in addition to scavenging of the OH radicals derived from water radiolysis, it was found that ascorbic acid promotes the chemical repair of precursors of AP-sites and base lesions more effectively than those of single strand breaks. We estimated the efficiency of the chemical repair of each lesion using a kinetic model. Approximately 50-60% of base lesions and AP-sites were repaired by 10 μM ascorbic acid, although strand breaks were largely unrepaired by ascorbic acid at low concentrations. The methods in this study will provide a route to understanding the mechanistic aspects of antioxidant activity in living systems.

A novel technique using DNA denaturation to detect multiply induced single-strand breaks in a hydrated plasmid DNA molecule by X-ray and4He2+ ion irradiation

To detect multiple single-strand breaks (SSBs) produced in plasmid DNA molecules by direct energy deposition from radiation tracks, we have developed a novel technique using DNA denaturation by which irradiated DNA is analysed as single-strand DNA (SS-DNA). The multiple SSBs that arise in both strands of DNA, but do not induce a double-strand break, are quantified as loss of SS-DNA using agarose gel electrophoresis. We have applied this method to X-ray and (4)He(2+) ion-irradiated samples of fully hydrated pUC18 plasmid DNA. The fractions of both SS-DNA and closed circular DNA (CC-DNA) exponentially decrease with the increasing dose of X rays and (4)He(2+) ions. The efficiency of the loss of SS-DNA was half that of CC-DNA for both types of irradiation, indicating that one of two strands in DNA is not broken when one SSB is produced in CC-DNA by irradiation. Contrary to our initial expectation, these results indicate that SSBs are not multiply induced even by high linear energy transfer radiation distributed in both strands.

Induction of genetic instability by transfer of a UV-A-irradiated chromosome

Exposure of cells to ultraviolet (UV)-A radiation induces oxidative damage in DNA, such as 8-oxo-7,8-dihydroguanine (8-oxoG), single-strand breaks, a-basic sites, and DNA-protein cross-links, via reactions with reactive oxygen species (ROS). In this study we examine whether the damage other than double-strand breaks (non-DSB damage), which is UV-A-induced oxidative damage, plays a role in the induction of chromosomal instability. We exposed human chromosome 21 to UV-A and transferred the chromosome into non-irradiated mouse recipient cells by microcell-mediated chromosome transfer. The chromosomal instability of both the transferred human chromosome and the recipient mouse chromosomes was examined by whole-chromosome painting and fluorescence in situ hybridization (WCP-FISH). The ploidy of the mouse recipient cells increased, and chromosomal aberrations occurred not only in the UV-A-irradiated human chromosome but also in the non-irradiated mouse chromosomes. The frequencies of these abnormalities increased with the radiation dose received by the transferred human chromosome. In contrast, in the control experiment, in which an non-irradiated human chromosome was transferred, the micro-cell hybrids remained diploid, and the frequency of chromosomal aberrations in both the transferred human chromosome and recipient mouse chromosomes remained low. Thus, the present study indicates that a chromosome harboring non-DSB damage induced by UV-A irradiation is unstable and transmits instability to chromosomes of non-irradiated recipient mouse cells.

Chemical repair activity of free radical scavenger edaravone: reduction reactions with dGMP hydroxyl radical adducts and suppression of base lesions and AP sites on irradiated plasmid DNA

Reactions of edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) with deoxyguanosine monophosphate (dGMP) hydroxyl radical adducts were investigated by pulse radiolysis technique. Edaravone was found to reduce the dGMP hydroxyl radical adducts through electron transfer reactions. The rate constants of the reactions were greater than 4 × 108 dm3 mol−1 s−1 and similar to those of the reactions of ascorbic acid, which is a representative antioxidant. Yields of single-strand breaks, base lesions, and abasic sites produced in pUC18 plasmid DNA by gamma ray irradiation in the presence of low concentrations (10–1000 μmol dm−3) of edaravone were also quantified, and the chemical repair activity of edaravone was estimated by a method recently developed by the authors. By comparing suppression efficiencies to the induction of each DNA lesion, it was found that base lesions and abasic sites were suppressed by the chemical repair activity of edaravone, although the suppression of single-strand breaks was not very effective. This phenomenon was attributed to the chemical repair activity of edaravone toward base lesions and abasic sites. However, the chemical repair activity of edaravone for base lesions was lower than that of ascorbic acid.