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A Simplified Cluster Analysis of Electron Track Structure for Estimating Complex DNA Damage Yields

松谷 悠佑; 中野 敏彰*; 甲斐 健師; 鹿園 直哉*; 赤松 憲*; 吉井 勇治*; 佐藤 達彦

International Journal of Molecular Sciences (Internet), 21(5), p.1701_1 - 1701_13, 2020/03

 被引用回数:13 パーセンタイル:73.17(Biochemistry & Molecular Biology)



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

端 邦樹; 漆原 あゆみ*; 山下 真一*; Lin, M.*; 室屋 裕佐*; 鹿園 直哉; 横谷 明徳; Fu, H.*; 勝村 庸介*

Journal of Radiation Research, 56(1), p.59 - 66, 2015/01


 被引用回数:6 パーセンタイル:31.67(Biology)

Reactions of edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) toward 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 higher than 4 $$times$$ 10$$^{8}$$ dm$$^{3}$$ 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 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 is 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.


Localization estimation of ionizing radiation-induced abasic sites in DNA in the solid state using fluorescence resonance energy transfer

赤松 憲; 鹿園 直哉; 齊藤 毅*

Radiation Research, 183(1), p.105 - 113, 2015/01

 被引用回数:19 パーセンタイル:67.29(Biology)

DNAに生じた傷(DNA損傷)のほとんどは生物が有する損傷修復機構によって元通りに修復されるが、中には修復困難なタイプの傷があり、これが突然変異や発癌の原因になるといわれている。修復困難とされる損傷型のひとつに「クラスター損傷」(複数の損傷がDNA鎖上の狭い領域に集中的に生じている)がある。しかしながら、その化学構造・損傷局在性の程度・生成頻度等の実体はほとんど明らかになっていない。そこで我々は、クラスター損傷の実体、特に局在性を実験的に解明するために、蛍光共鳴エネルギー移動を利用した損傷位置局在性評価法(FRET法)の開発を行ってきた。検出対象の損傷には脱塩基部位(AP)を選び、FRET法を、種々の放射線($$^{60}$$Co $$gamma$$線,ヘリウム・炭素イオンビーム)を照射した乾燥DNA試料に適用したところ、特に飛跡末端の炭素イオンビームでは、飛跡中でクラスター化した損傷が生じることが明らかとなった。


Towards laser driven hadron cancer radiotherapy; A Review of progress

Ledingham, K.*; Bolton, P. R.*; 鹿園 直哉; Ma, C.-M.*

Applied Sciences (Internet), 4(3), p.402 - 443, 2014/09

 被引用回数:75 パーセンタイル:88.44(Chemistry, Multidisciplinary)

It has been known for about sixty years that proton and heavy ion therapy is a very powerful radiation procedure for treating tumors. It has an innate ability to irradiate tumors with greater doses and spatial selectivity compared with electron and photon therapy and, hence, is a tissue sparing procedure. For more than twenty years, powerful lasers have generated high energy beams of protons and heavy ions and it has, therefore, frequently been speculated that lasers could be used as an alternative to radiofrequency (RF) accelerators to produce the particle beams necessary for cancer therapy. The present paper reviews the progress made towards laser driven hadron cancer therapy and what has still to be accomplished to realize its inherent enormous potential.


Order effect of repair processes to clustered DNA damage

白石 伊世; 鈴木 雅雄*; 鹿園 直哉; 藤井 健太郎; 横谷 明徳

Journal of Radiation Research, 55(Suppl.1), p.i92 - i93, 2014/03

In a living cell, cluster DNA damage is thought to be processed by several different pathways simultaneously or sequentially. Under this situation the cellular response to cluster DNA might depend on the order of repair processes because the configuration of the lesions will be modified by the reaction of initial repair protein, affecting the DNA-binding or excision activities of latter proteins. In the present study, we investigate whether initial enzymatic repair affects latter processes. Plasmid DNA exposed to C ion is treated with two base excision repair enzymes, Nth and Fpg, which convert pyrimidine and purine lesions to a SSB, respectively. Obtained results show that the amount of enzymatically induced SSB is very slightly less in DNA sample treated with Nth first and then Fpg than that of other treatments. These results indicate that the configuration-change of the cluster by the first enzymatic treatment does not significantly influence the activity of secondary enzyme.


Significance of DNA Polymerase I in ${it in vivo}$ processing of clustered DNA damage

鹿園 直哉; 赤松 憲; 高橋 桃子*; 野口 実穂; 漆原 あゆみ; O'Neill, P.*; 横谷 明徳

Mutation Research; Fundamental and Molecular Mechanisms of Mutagenesis, 749(1-2), p.9 - 15, 2013/09

 被引用回数:12 パーセンタイル:39.04(Biotechnology & Applied Microbiology)

クラスターDNA損傷は、電離放射線によってDNAへリックス二回転中に二つ以上の損傷が生じるものである。クラスターDNA損傷がどの程度、また、どのように生物影響を及ぼすのかに関しては不明な点が多い。本研究では、鎖切断と脱塩基部位や8-oxo-7,8-dihydroguanine (8-oxoG)を含むクラスターDNA損傷を用い、大腸菌に形質転換し、形質転換効率及び突然変異頻度を調べた。鎖切断と脱塩基部位からなるクラスターDNA損傷の場合、鎖切断及び脱塩基部位がそれぞれ単独であった場合に対し、形質転換効率は大幅に低下することが明らかになった。損傷間の距離を離す(10-20bp)と、形質転換効率はDNA polymerase I(Pol I)の作用により回復した。一方、鎖切断と8-oxoGからなるクラスターDNA損傷の場合、クラスターDNA損傷による突然変異頻度はPol Iの働きによって低下することが明らかとなった。これらの結果は、クラスターDNA損傷による生物効果にPol Iが深く関与することを示している。


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

端 邦樹; 漆原 あゆみ; 山下 真一; 鹿園 直哉; 横谷 明徳; 勝村 庸介*

Biochemical and Biophysical Research Communications, 434(2), p.341 - 345, 2013/05

 被引用回数:8 パーセンタイル:25.42(Biochemistry & Molecular Biology)

In order to clarify whether ascorbic acid, which is a major antioxidant in living systems, chemically repairs radiation damage to DNA. We quantified the yields of base lesions, AP sites and single strand breaks (SSBs) produced in plasmid DNA by $$gamma$$-irradiation in the presence of ascorbic acid with concentrations of 10-100 $$mu$$MM. By comparing the suppression efficiencies to the induction of each DNA lesion, 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 $$mu$$M 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 Methodology for estimating localization of apurinic/apyrimidinic sites in DNA using fluorescence resonance energy transfer

赤松 憲; 鹿園 直哉

Analytical Biochemistry, 433(2), p.171 - 180, 2013/02

 被引用回数:5 パーセンタイル:19.76(Biochemical Research Methods)

We have developed a methodology for estimating localization of lesions on double-stranded DNA using fluorescence resonance energy transfer (FRET). We have focused on apurinic/apyrimidinic (AP) sites, which are typical DNA lesions induced by radiation and chemicals, and produced spontaneously under physiological conditions. Donor-acceptor fluorescent probes with $$O$$-amino groups (AlexaFluor 350-488 dye pair) were used for selectively labeling AP sites. PUC19 plasmid subjected to heat treatment was used as a model double-stranded DNA containing AP sites. The results of both FRET analysis and theoretical study enabled us to prove that AP sites induced by the heat treatment are distributed almost randomly along the DNA molecule. This methodology will be useful for estimating the risk of ionizing radiation and chemicals based on the probability of producing "clustered DNA damage sites", which are considered to be less easily repairable and therefore more harmful to living systems.


Yield of single- and double-strand breaks and nucleobase lesions in fully hydrated plasmid DNA films irradiated with high-LET charged particles

牛込 剛史*; 鹿園 直哉; 藤井 健太郎; 渡辺 立子; 鈴木 雅雄*; 鶴岡 千鶴*; 田内 広*; 横谷 明徳

Radiation Research, 177(5), p.614 - 627, 2012/05

 被引用回数:21 パーセンタイル:68.02(Biology)

イオンビーム照射したDNA損傷のLET依存性を明らかにするため、水和DNAフィルム中に誘発されるDNA損傷収率を調べた。1本鎖切断収率はHe, C及びNeイオンのLETの増加とともに減少したのに対して、2本鎖切断収率は増加した。照射DNAフィルムを塩基除去修復酵素で処理すると、酵素の作用により鎖切断が生じたことから照射により水和DNAフィルム中にも塩基損傷が生じることがわかった。しかしその収率はLETの増加とともに顕著に現象したことから、高LETほど難修復性の複雑なDNA損傷を誘発することが推測された。


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

野口 実穂; 漆原 あゆみ; 横谷 明徳; O'Neill, P.*; 鹿園 直哉

Mutation Research; Fundamental and Molecular Mechanisms of Mutagenesis, 732(1-2), p.34 - 42, 2012/04

 被引用回数:16 パーセンタイル:47.05(Biotechnology & Applied Microbiology)

In vivoにおいて塩基損傷を伴った鎖切断の効果はいまだ多くが不明である。本研究においてわれわれは1ヌクレオチドgap(GAP)と8-oxoGから成るクラスターDNA損傷の突然変異誘発性について明らかにした。MutY欠損株において、GAPと8-oxoGの2つの損傷から成り、これらが相補鎖にまたがって存在するクラスター損傷の変異頻度は8-oxoGとAPサイトから成るクラスター損傷とほぼ同じ値を示したことから、クラスター損傷内において、GAPはAPサイトと同様に処理されることが示唆された。GAPと8-oxoGが同一鎖上に存在するクラスター損傷の変異頻度は8-oxoG単独損傷とほぼ等しい値であった。3つの損傷から成るクラスター損傷(GAPと8-oxoGが相補鎖、その同一鎖上にもう一つの8-oxoG)の変異頻度は8-oxoG単独損傷より高い値であったが、2つの8-oxoGが相補鎖にまたがって存在するクラスター損傷に比べて低い値であった。これらの結果から、クラスター損傷内にGAPが存在する場合、存在しない場合に比べて、8-oxoGの鋳型として向いに挿入されるヌクレオチドの変異誘発性が低くなることが示唆された。さらに、GAPの修復は相補鎖の8-oxoGにより阻害されるが、同一鎖の8-oxoGには阻害されないことが示唆され、GAPの修復が生物影響に大きく影響を与えることが示された。


A Novel technique using DNA denatureation to detect multiplly induced single-strand breaks in a hydrated plasmid DNA molecule by X-ray and $$^{4}$$He$$^{2+}$$ ion irradiation

横谷 明徳; 鹿園 直哉; 藤井 健太郎; 野口 実穂; 漆原 あゆみ

Radiation Protection Dosimetry, 143(2-4), p.219 - 225, 2011/02

 被引用回数:3 パーセンタイル:26.82(Environmental Sciences)



A Model for analysis of the yield and the level of clustering of radiation-induced DNA-strand breaks in hydrated plasmids

鹿園 直哉; 横谷 明徳; 漆原 あゆみ; 野口 実穂; 藤井 健太郎

Radiation Protection Dosimetry, 143(2-4), p.181 - 185, 2011/02

 被引用回数:2 パーセンタイル:18.99(Environmental Sciences)

Although it is widely accepted that the spatial distribution of strand breaks is highly relevant to the biological consequences of radiation, the extent to which strand breaks are clustered is not usually demonstrated directly from experimental data. To evaluate the spatial distribution of radiation-induced strand breaks, we have developed a model for the generation of strand breaks after irradiation. To find out whether the model is valid, we compared the calculated values to experimental data obtained by a plasmid DNA assay. Taking into account the inherent bias of the plasmid assay, the model described well the experimental results of hydrated plasmids exposed to radiation.


Spectroscopic study of radiation-induced DNA lesions and their susceptibility to enzymatic repair

横谷 明徳; 藤井 健太郎; 鹿園 直哉; 鵜飼 正敏*

Charged Particle and Photon Interactions with Matter; Recent Advances, Applications, and Interfaces, p.543 - 574, 2010/12

このレヴューは、「荷電粒子及び光子と物質との相互作用; 最近の進展,応用及び境界領域」について書かれた本の一章である。このレヴューでは、われわれが最近放射光やイオンビームを用いて研究を行ったDNA変異に関する最新の知見を紹介するとともに、DNA変異に対する酵素的修復作用についての生化学的及び大腸菌を使った突然変異誘発に関する成果を示し、過去の知見と比較しながらDNA変異の生物応答に果たす役割を論じる。


Protective effects of silybin and analogues against X-ray radiation-induced damage

Fu, H. Y.*; Lin, M.; 勝村 庸介*; 横谷 明徳; 端 邦樹; 室屋 裕佐*; 藤井 健太郎; 鹿園 直哉

Acta Biochimica et Biophysica Sinica, 42(7), p.489 - 495, 2010/07

 被引用回数:12 パーセンタイル:32.21(Biochemistry & Molecular Biology)

Silybin(SLB)及びその類似体であるhesperetin(HESP), naringenin(NAN), naringin(NAR)は化学抗ガン剤として報告されている天然フラボノイドの有効成分であると考えられている。また、これらの物質がDNA塩基の酸化損傷を化学的に修復することをパルスラジオリシス法によって明らかにしてきた。そこで本研究では、in vitroにおいてプラスミドDNAの軟X線誘起損傷に対するSLBとその類似体による保護効果を調べた。X線照射の結果、保護効果を調べた物質の中ではHESPとSLBがX線によって誘起されるDNA一本鎖切断(SSB)を最も効果的に抑制することがわかった。さらに他の実験結果と比較すると、SLBやその類似体がin vitroでDNA損傷を抑制する能力はそれらのフリーラジカル捕捉能と関連があることがわかった。以上により、天然フラボノイド,SLB及びその類似体が放射線損傷に対する保護剤として有効に働くことを明らかにした。


Studies on biological effects of ion beams on lethality, molecular nature of mutation, mutation rate, and spectrum of mutation phenotype for mutation breeding in higher plants

田中 淳; 鹿園 直哉; 長谷 純宏

Journal of Radiation Research, 51(3), p.223 - 233, 2010/05

 被引用回数:164 パーセンタイル:96.1(Biology)

Recently, heavy ions or ion beams have been used to generate new mutants or varieties, especially in higher plants. It has been found that ion beams show high relative biological effectiveness (RBE) of growth inhibition, lethality, and so on, but the characteristics of ion beams on mutation have not been clearly elucidated. To understand the effect of ion beams on mutation induction, mutation rates were investigated using visible known Arabidopsis mutant phenotypes, indicating that mutation frequencies induced by carbon ions were 20-fold higher than by electrons. In chrysanthemum and carnation, flower-color and flower-form mutants, which are hardly produced by $$gamma$$-rays or X-rays, were induced by ion beams. Novel mutants and their responsible genes, such as UV-B resistant, serrated petals and sepals, anthocyaninless, etc. were induced by ion beams. These results indicated that the characteristics of ion beams for mutation induction are high mutation frequency and broad mutation spectrum and therefore, efficient induction of novel mutants. On the other hand, PCR and sequencing analyses showed that half of all mutants induced by ion beams possessed large DNA alterations, while the rest had point-like mutations. Both mutations induced by ion beams had a common feature that deletion of several bases were predominantly induced. It is plausible that ion beams induce a limited amount of large and irreparable DNA damage, resulting in production of a null mutation that shows a new mutant phenotype.


Nucleobase lesions and strand breaks in dry DNA thin film selectively induced by monochromatic soft X-rays

藤井 健太郎; 鹿園 直哉; 横谷 明徳

Journal of Physical Chemistry B, 113(49), p.16007 - 16015, 2009/11

 被引用回数:19 パーセンタイル:44.43(Chemistry, Physical)



Biological consequences of potential repair intermediates of clustered base damage site in Escherichia coli

鹿園 直哉; O'Neill, P.*

Mutation Research; Fundamental and Molecular Mechanisms of Mutagenesis, 669(1-2), p.162 - 168, 2009/11

 被引用回数:17 パーセンタイル:45.87(Biotechnology & Applied Microbiology)

To gain insights into the processing of bistranded base lesions, several potential repair intermediates following 8-oxoG removal from bistranded clusters containing 8-oxoG and DHT were assessed. Clusters, such as DHT/AP and DHT/GAP have relatively low mutation frequencies, whereas clusters, such as AP/AP or GAP/AP, significantly reduce the number of transformed colonies, most probably through formation of a lethal DSB. These results suggest that bistranded base lesions, but not clusters containing only AP sites and strand breaks, are repaired in a coordinated manner so that the formation of DSBs is avoided. We propose that, when either base lesion is initially excised from a bistranded base damage site, the remaining base lesion will only rarely be converted into an AP site or a single strand break in vivo.


Free radical scavenging reactions and antioxidant activities of silybin; Mechanistic aspects and pulse radiolytic studies

Fu, H. Y.*; Lin, M.; 室屋 裕佐*; 端 邦樹; 勝村 庸介; 横谷 明徳; 鹿園 直哉; 籏野 嘉彦

Free Radical Research, 43(9), p.887 - 897, 2009/09

 被引用回数:24 パーセンタイル:48.33(Biochemistry & Molecular Biology)

Silybin (extracted from Silybum marianum) is the major active constituent of silymarin which possesses a wide range of medicinal properties. These properties may be, in part, due to the potent scavenging capacity of oxidizing free radicals. In this context, scavenging radicals (hydroxyl, azide, dibromide anion radicals, nitrite, carbonate, etc.) of silybin have been studied to understand the mechanistic aspects of its action against free radicals. The transients produced in these reactions have been assigned and the rate constants have been measured by pulse radiolysis techniques. Reduction potential determined both by cyclic voltammetry gave a value 0.62$$pm$$0.02 V vs NHE at pH 9. Quantum chemical calculations have been performed to further confirm the different activities of individual hydroxyl groups with the difference of heat of formation. Moreover, silybin also protected plasmid pUC18 DNA from soft X-ray radiation which induced strand breaks. These results are expected to be helpful for a better understanding of the anti-oxidative properties of silybin.


Processing of thymine glycol in a clustered DNA damage site; Mutagenic or cytotoxic

Bellon, S.*; 鹿園 直哉; Cunniffe, S. M. T.*; Lomax, M.*; O'Neill, P.*

Nucleic Acids Research, 37(13), p.4430 - 4440, 2009/07

 被引用回数:45 パーセンタイル:70.27(Biochemistry & Molecular Biology)

The potential for genetic change arising from the effects of clustered damage sites is high. We have investigated whether clusters containing thymine glycol (Tg) are highly mutagenic or lead to potentially cytotoxic lesions, when closely opposed to either 8-oxo-7,8-dihydroguanine (8-oxoG) or an AP site. We have shown that double strand breaks (DSBs) arise when Tg is opposite to an AP site, either through attempted repair or at replication. In contrast, 8-oxoG opposite to Tg in a cluster protects against DSB formation but does enhance the mutation frequency at the site of 8-oxoG relative to that at a single 8-oxoG.


A UVB-hypersensitive mutant in ${it Arabidopsis thaliana}$ is defective in the DNA damage response

坂本 綾子; Lan, V. T. T.*; Puripunyavanich, V.*; 長谷 純宏; 横田 裕一郎; 鹿園 直哉; 中川 繭*; 鳴海 一成; 田中 淳

Plant Journal, 60(3), p.509 - 517, 2009/07

 被引用回数:20 パーセンタイル:52.33(Plant Sciences)

細胞分裂周期の進行は、内的又は外的なさまざまな要因によって常にストレスを受けており、それによってDNA複製の中断や染色体の分配異常などが引き起こされることが知られている。細胞周期チェックポイントとは、こうした異常事態の際に細胞周期の進行を停止し、適切な処理が行われるまで次のステップに移行しないようにするための機構である。われわれは、シロイヌナズナの紫外線感受性変異株のスクリーニングの過程で、${it suv2}$ (sensitive to UV 2)変異株を単離した。${it suv2}$変異株は、さまざまなDNA変異原やヒドロキシウレアに対して感受性を示し、その表現系は損傷チェックポイントにかかわるAtATRの欠損株に非常によく似ていた。さらに、SUV2蛋白質はGCN4型のコイルドコイルドメインを持つ蛋白質をコードしており、N末側にはPI3K様プロテインキナーゼのターゲット配列が2か所存在していた。そこで、SUV2蛋白質同士どうしの相互作用を酵母two-hybridの系で解析したところ、${it suv2}$は2量体を形成することが明らかになった。以上の結果から、われわれは${it suv2}$がATRの活性を制御するATRIPのシロイヌナズナにおけるホモログであると結論づけ、AtATRIPと命名した。

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