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椎名 卓也; 菅谷 雄基; 白石 伊世; 渡辺 立子; 鈴木 雅雄*; 横谷 明徳
no journal, ,
今まで明らかにされていなかったAPサイト及びAPクラスターの誘発過程と炭素イオン(290MeV/n, LET13, 60keV/um)、あるいはX線のトラック構造との関係を明らかにするため、放射線照射したpUC18プラスミドDNAの酵素処理により放射線誘発性APサイトの収率を定量した。幾つかのスキャベンジャー能のサンプルでAPサイトの誘発について拡散性OHラジカルの間接効果を評価した。その結果、これまでの研究で明らかにされていた鎖切断や塩基損傷と同様のスキャベンジャー能依存性を示した。さらに、調べた放射線のすべてでAPサイトと鎖切断のスキャベンジャー能依存性の挙動が一致したことから、鎖切断とAPサイトが同様の生成過程を経ており、塩基損傷の生成過程とは異なることが示唆された。この結果をモンテカルロシミュレーションから得られた損傷収率と比較,検討する予定である。
菅谷 雄基; 椎名 卓也; 白石 伊世; 藤井 健太郎; 横谷 明徳
no journal, ,
酸素のK殻イオン化を引き起こすと、塩基損傷収率が窒素のK殼イオン化の収率と比べ急激に増えることがわかっている。本研究では、軟X線を照射したDNAに生成される鎖切断や塩基損傷,APサイトを定量し、内殻励起やイオン化が選択的損傷生成に果たす役割を明らかにすることを目的とする。試料にはプラスミドDNAを用い、SPring-8のBL23SUを使用して照射を行った。ピリミジン塩基損傷及びプリン塩基損傷,APサイトの検出は、それぞれNth, Fpg, Nfoの3種類のDNAグリコシレースで処理しSSB(single strand break)に変え、アガロース電気泳動法によって定量した。その結果、予想に反しDNA中の酸素を1sから
へ選択的に励起を引き起こす軟X線を照射すると、DNA損傷収率が大きく減少することがわかった。これは、励起により生じた1価の陽イオンは、イオン化による2価の陽イオンに比べ、損傷誘発に大きくは寄与しない可能性を示している。本国際会議では、酸素のK殼イオン化エネルギー領域についてのDNA損傷の収率変化を報告し、それらがDNA損傷の選択的な生成機構に寄与するかについて議論する。
鹿園 直哉; 野口 実穂; 漆原 あゆみ; O'Neill, P.*; 横谷 明徳
no journal, ,
Clustered DNA damage, defined as two or more lesions within one to two helical turns of DNA induced by a single radiation track, is a unique feature of ionizing radiation. To examine the role of strand break repair in mutation induction, the clustered single strand break (SSB) + 8-oxo-7,8-dihydroguanine (8-oxoG) lesions were transfected in the 
strain of 
, which lacks Pol I that participates in repair synthesis. We found that the mutation frequencies of SSB + 8-oxoG lesions become markedly higher in the strain than those in 
strain, while the mutation frequencies of single lesions remained low in . These results indicate that Pol I plays an important role in protecting against mutation and suggest that the extent of strand break repair determines the mutagenic potential of bi-stranded clustered DNA damage containing base lesions.
白石 伊世; 椎名 卓也; 菅谷 雄基; 鹿園 直哉; 横谷 明徳
no journal, ,
In a living cell, a multiply damaged site in DNA is thought to be processed by several different pathways simultaneously or sequentially. Under this situation the cellular response to the lesion cluster 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 lesion-excision activities of latter repair proteins. Plasmid DNA (pUC18) irradiated with 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 configurational change of the cluster by the first enzymatic treatment does not significantly influence the activity of secondary enzyme.
漆原 あゆみ; 児玉 靖司*; 横谷 明徳
no journal, ,
It is well known that ionizing radiation induces genetic instability in the progeny of irradiated cells. Since it seems unlikely that DNA double-strand breaks (DSBs) persist through several cycles of cell division, we hypothesize that some DNA lesions, but not DSBs per se, are also involved in inducing genetic instability. To find out whether genetic instability is induced by non-DSBs type of damage, particularly oxidative base lesions, we transferred a human chromosome exposed to UV-A into unirradiated mouse recipient cells by microcell fusion, and examined chromosome aberrations of the transferred human chromosome and recipient mouse chromosomes. The transferred chromosomes were analyzed by the whole chromosome painting fluorescence in situ hybridization. Most of the microcell hybrids transferred with the unirradiated human chromosomes remained diploid, and both the transferred human chromosome and recipient mouse chromosomes were stable. In contrast, the microcell hybrids transferred with the UV-A irradiated human chromosome became polyploid and structural aberrations occurred not only in the UV-A irradiated human chromosome but also in the unirradiated mouse chromosomes. These results suggest that non-DSBs type of DNA damage induced by UV-A irradiation disturbs the homeostatic maintenance of the chromosomes and induces genetic instability.
rster resonance energy transfer赤松 憲; 鹿園 直哉
no journal, ,
DNA lesions induced by ionizing radiation and chemicals can cause mutation and carcinogenesis. In particular, "clustered damage" site, that is a DNA region with multiple lesions within a few helical turns, is believed to hardly be repaired. This type of damage is considered to be induced around high-LET radiation tracks. However, chemical and spatial details of them are not known. Therefore, we have developed a methodology for estimating the degree of localization of the lesions using Frster resonance energy transfer (FRET). First, we prepared pUC19 with APsites (AP-DNA) by heating in acidic buffer as a randomly-distributed AP-DNA model. The AP-DNA was labeled both with Alexa Fluor 350 and 488 fluorescent dyes (donor and acceptor) with -O-NH
groups. The FRET efficiency (E) was calculated using the donor intensities before/after enzymatic digestion of the labeled AP-DNA. Moreover, we have constructed a theoretical equation to obtain E as functions both of AP-site density and of labeling ratio of acceptor to total AP-sites. As a result, we found that experimentally-obtained E values for the heat-treated AP-DNA correspond to theoretical ones calculated on the basis of exponential distribution. Now we are applying the FRET methodology to the DNA irradiated with radiations such as 
Co 
-rays and helium ion beam.
渡辺 立子; 椎名 卓也*; 横谷 明徳
no journal, ,
生体が放射線照射されて生じるDNA損傷は、そのほとんどは修復機能により修復されるが、中には、完全に修復されずに残ってしまう損傷があり、細胞死や突然変異などの原因となると考えられる。その実態は完全に明らかにされてはいないが、DNA2本鎖切断の一部に加えて、鎖切断と塩基損傷がごく近傍に生じたようなnon-DSBタイプのクラスター損傷も重要であるとされている。われわれは、DNA損傷生成過程のモデル化とシミュレーション計算によりさまざまな放射線によるDNA損傷のスペクトルを推定してきた。本発表では、より精度の高いシミュレーションを目指し、最近蓄積しつつあるnon-DSBタイプのクラスター損傷の実験データを考慮に入れ、塩基損傷の生成モデルについて検討した結果を示す。その結果を反映し、さまざまな放射線に対するDNA損傷スペクトルをLETだけではなく、マイクロドジメトリ量を指標として解析する。