Ken Akamatsu

X-ray absorption near edge structure of DNA bases around oxygen and nitrogen K-edge

Abstract X-ray absorption near edge structure (XANES) spectra of DNA and the nucleobases (adenine, guanine, cytosine, thymine and uracil) are observed using monochromatic soft X-rays (400–560 eV). The photon energy range includes the electron binding energy of the core orbital of nitrogen and oxygen K shell. Obtained XANES spectra showing a characteristic structure were theoretically analyzed by the discrete variational density functional formalism (DV-Xα) method. These results clearly indicate that the photo-excitation of oxygen and nitrogen 1s electron to π* orbitals shows a sharp resonance profile rather than that to σ* orbitals, enough to realize selecting a photochemical reaction site at specific nucleobases in a DNA molecule. Novel experiments are proposed based on the obtained spectra to understand the molecular mechanisms of radiation damages on DNA bases.

X-ray absorption near edge structures of DNA or its components around the oxygen K-shell edge.

Abstract Akamatsu, K. and Yokoya, A. X-Ray Absorption near Edge Structures of DNA or its Components around the Oxygen K-shell Edge. The initial process of radiation damage in DNA was investigated by measuring the X-ray absorption near edge structures (XANES) within the energy region around the oxygen K-shell absorption edge for DNA, cytosine and 2-deoxy-d-ribose. Irradiation and XANES experiments were performed with the BL23SU soft X-ray beamline, using synchrotron radiation from the 8 GeV electron storage ring at SPring-8. Samples were mounted on gold-coated plates in a vacuum chamber. The XANES spectra were obtained by measuring the photoelectron current of the samples. 2-Deoxy-d-ribose was exposed to X rays at the absorption peak corresponding to the oxygen (O) 1s→σ* transition energy (538 eV); the XANES spectra were obtained after each irradiation. DNA and cytosine, possessing characteristic XANES spectra, both had two major energy bands corresponding to the O 1s→π* and 1s→σ* transitions. Two new peaks appeared and gradually increased in the XANES spectra of 2-deoxy-d-ribose during irradiation. These results suggest that C–O bonds in 2-deoxy-d-ribose are transformed to C=O bonds by O 1s→σ* transition, suggesting that the molecules undergo chemical changes into carbonyl-containing compounds.

Localization Estimation of Ionizing Radiation-Induced Abasic Sites in DNA in the Solid State Using Fluorescence Resonance Energy Transfer

Clustered DNA damage is considered an important factor in determining the biological consequences of ionizing radiation. In this study, we successfully estimated the localization of abasic sites (APs) in DNA exposed to ionizing radiation using fluorescence resonance energy transfer (FRET) without any involvement of repair enzyme functions. A linearized plasmid (pUC19 digested by Sma I) was irradiated with: 60Co γ rays; 4He2+ (2.0 MeV/u) particles; and the 12C5+ (0.37 MeV/u) particles in the solid state. A donor or acceptor fluorescent probe with a nucleophilic O-amino group was used to label APs. The results showed that the 12C5+ particle likely produced close APs within a track. The apparent distance calculated from the observed FRET efficiency (E) of around 0.10 was estimated to be approximately 17 base pairs. On the other hand, E values of 60Co γ rays and the 4He2+ beam were less than those of the 12C5+ beam, increased with increasing AP density (the average number of APs per base pair), and were slightly greater than those of randomly distributed APs. We propose that the FRET method provides a degree of localization regardless of whether an AP cluster is single-stranded or bistranded DNA damage.

Ion Desorption from DNA Components Irradiated with 0.5 keV Ultrasoft X-Ray Photons

Abstract Fujii, K., Akamatsu, K. and Yokoya, A. Ion Desorption from DNA Components by Ultrasoft X-Ray Photons. Radiat. Res. 161, 435–441 (2004). Positive ion desorption from thin films of DNA components, 2-deoxy-d-ribose, thymine, thymidine (dThd), and thymidine 5′-monophosphate (dTMP) was investigated in the oxygen K- shell edge excitation region using synchrotron ultrasoft X rays (538 eV). A large number of molecular fragments, H+, CHx+, C2Hx+, CO+, CHxO+, C3Hx+, C2HxO+ and C3HxO+ (x = 1, 2 and 3), were observed as desorbed ions from 2-deoxy-d-ribose. Some of these ions are related to simultaneous bond scission at particular C–C and C–O (or C–C) bonds in the furanose ring structure in the 2-deoxy-d-ribose molecule, indicating that the impact of photons on the oxygen atom and the impact of ejected secondary electrons (e.g. Auger electrons) cause an intense destruction of the furanose ring structure. In thymine thin films, H+, CHx+, CO+, CHxO+, C2HxN+ and CHxNO+ (x = 1, 2 and 3) fragments were observed. The yields of these ions were smaller than the yields from 2-deoxy-d-ribose. The desorption of CH3+ from thymine might induce a molecular conversion from thymine to uracil. The mass patterns of dThd and dTMP, and especially that of dTMP, were similar to that of 2-deoxy-d-ribose, indicating that a number of ions were generated at the sugar site, even in the nucleotide molecule. It is therefore predicted that the sugar moiety is more fragile than the thymine base.

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.

Qualitative and Quantitative Analyses of the Decomposition Products that Arise from the Exposure of Thymine to Monochromatic Ultrasoft X Rays and 60Co Gamma Rays in the Solid State

Abstract Akamatsu, K., Fujii, K. and Yokoya, A. Qualitative and Quantitative Analyses of the Decomposition Products that Arise from the Exposure of Thymine to Monochromatic Ultrasoft X Rays and 60Co Gamma Rays in the Solid State. Radiat. Res. 161, 442–450 (2004). HPLC analyses of condensed thymine irradiated with monochromatic synchrotron ultrasoft X rays in the energy region around nitrogen and oxygen K-shell edges were performed. Cobalt-60 γ rays were used as a reference radiation. The radiation chemical dose response of each separated thymine decomposition product was also determined. Uracil (U), 5-(hydroxymethyl)uracil (HMU), 5,6-dihydrothymine (DHT), 5-formyluracil (foU) and four main unknown products were found in the HPLC chromatogram of the sample irradiated with ultrasoft X rays in vacuo. Similar spectra of the products were also found in the γ-ray experiment; however, some unknown products that appeared after elution of the thymine peak were significantly larger than those in the ultrasoft X- ray experiment. This result indicates the difference in radiation quality. The G value of DHT produced by γ radiation was 10 times larger than those produced by the ultrasoft X- ray photons with energies of 395 and 407 eV corresponding to below and on the nitrogen K-shell edge, respectively. This result suggests that the differences in the photon energy and/ or in the energy spectra of the secondary electron between ultrasoft X rays and γ rays are causing differences in the process of the radiation chemistry. Moreover, the yields of all the thymine decomposition products induced by 538 eV photons (oxygen K-shell edge) were significantly smaller than those induced by photons around the nitrogen K-shell edge. The K-shell excitation of oxygen in thymine may efficiently promote the production of small thymine fragments susceptible to desorption from the sample.

Decomposition of 2-deoxy-D-ribose by irradiation with 0.6 keV electrons and by 0.5 keV ultrasoft X-rays.

Purpose: To compare the molecular decomposition of 2‐deoxy‐D‐ribose induced by 0.6 keV electron irradiation or by 0.5 keV ultrasoft X‐ray irradiation. Materials and methods: A thin film of 2‐deoxy‐D‐ribose was irradiated by two radiation sources: low‐energy (∼0.6 keV) electrons and ultrasoft X‐rays (∼0.5 keV). The positive ions that were desorbed from the sample during the irradiation were measured using a quadrupole mass spectrometer. The spectral changes in the X‐ray absorption near edge structure (XANES) were also examined after the irradiation. Results and discussion: The ions that were desorbed from 2‐deoxy‐D‐ribose due to electron irradiation were mainly H+, CHx+, C2Hx+, CO+, CHxO+, C3Hx+, C2HxO+ and C3HxO+ (x=1, 2, and 3) ions. These ions were the same as those observed in desorption due to ultrasoft X‐ray irradiation. The XANES spectral changes induced by electron irradiation showed C‐O bond cleavage in the molecule and C=O bond formation in the surface residues. These results show that intensive molecular decomposition of the furanose ring structure was induced by both types of irradiation. It is inferred that these irradiation products are primarily produced by secondary electrons (several tens of eV), which are thought to be generated by both types of irradiation when they are applied to the 2‐deoxy‐D‐ribose sample.

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.

Low-energy Auger- and Photo-electron Effects on the Degradation of Thymine by Ultrasoft X-irradiation

Purpose: To investigate quantitatively and qualitatively the production of thymine radicals produced by monochromatic ultrasoft X (USX) ‐ or 60Co γ‐rays using electron paramagnetic resonance (EPR) spectroscopy. Materials and Methods: Thymine was chosen as the DNA component for the irradiation. The EPR experiments of irradiated thymine were performed using an X‐band EPR device installed in a soft X‐ray beamline (BL23SU) in SPring‐8. Sample pellets were irradiated with USX photons in a microwave cavity in a vacuum chamber. EPR measurements of thymine powder pellets irradiated with USX photons at energies of 407 and 538 eV were performed at 77 K or room temperature. For reference, 60Co γ‐irradiation to a pellet was also performed at room temperature. Results: The following three features were found: 1) comparison between the two energies shows that the EPR dose‐response curves are clearly distinguishable from each other: the curve for 407 eV saturated at a lower dose and spin number than that for 538 eV. 2) no evident qualitative difference between the radical species produced at the two energies was observed. 3) the EPR signal of the 538 eV USX‐irradiated sample measured after annealing for 12 days is similar to that obtained with 60Co γ‐irradiation. Conclusions: The difference observed in the EPR dose‐response relationship reflects the difference in the K‐absorption cross‐sections of carbon, nitrogen and oxygen in the thymine molecule which govern the photo‐/Auger electron energy spectrum.

Infrared spectral change in 2-deoxy-d-ribose by irradiation with monochromatic photons around oxygen K-edge

Abstract Analyses of chemical changes in DNA by energy deposition from ionizing radiation are quite important to strictly know characteristics of radiobiological effects. Monochromatic photons from synchrotron radiation are one of the powerful probes to investigate the effects. As a step for the aim, chemical analyses by Fourier-transform infrared spectroscopy of the samples irradiated with the monochromatic photons were performed. It appeared that 2-deoxy- d -ribose irradiated around the energy of oxygen K-edge contained CO or CC, which would be responsible for a direct strand break of DNA. These data are noteworthy to find not only the strand scission at 2-deoxy- d -ribose moiety by the direct energy deposition by photon but also the following radiobiological responses such as cell killing or mutation.