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Mutagenic potential of clustered DNA damage site in ${it Escherichia coli}$

大腸菌におけるクラスターDNA損傷の突然変異誘発性

鹿園 直哉; Pearson, C.*; Thacker, J.*; O'Neill, P.*

Shikazono, Naoya; Pearson, C.*; Thacker, J.*; O'Neill, P.*

クラスターDNA損傷は、電離放射線によって特異的に生じるものであるが、その変異誘発機構については不明な点が多い。そこで、二本鎖上の任意の位置に二つの塩基損傷(8-oxo-7,8-dihydroguanine (8-oxoG)及びdihydrothymine (DHT))を配置させ、単独の塩基損傷に比べて大腸菌において変異誘発頻度が高まるかどうかを調べた。その結果、野生株,グリコシラーゼ欠損株において、8-oxoGもしくはDHT単独に対し8-oxoGとDHTとがクラスター化することで突然変異頻度は高まることが見いだされた。本研究で用いたクラスターDNA損傷では、DHTが先に除去される結果、多くの8-oxoGの除去が阻害されると考えられるが、8-oxoGが先に除去される可能性も残っている。クラスター損傷の修復過程に関してさらなる知見を得る目的から、8-oxoGが除去された後に生ずると考えられる中間体の変異誘発頻度を調べた。その結果、「DHTとAP」もしくは「DHTと鎖切断」では変異頻度が高くないが、「APとAP」もしくは「APと鎖切断」では変異頻度が非常に高いことがわかった。これらの結果から、8-oxoGもしくはDHTが除去された後に残ったクラスター損傷内の塩基損傷は、AP部位や1本鎖切断には変換されないことが示唆された。

Clustered DNA damage induced by a single radiation track is a unique feature of ionizing radiation. Recent ${it in vitro}$ studies have shown that the repair of lesions within clusters may be retarded, but less is known about the processing and the mutagenic effects of such clustered damage in vivo. Using a bacterial plasmid-based assay, we have investigated the mutagenic potential of bistranded clustered damage sites which consist of 8-oxo-7,8-dihydroguanine (8-oxoG) and dihydrothymine (DHT) at defined separations. We found a significantly higher mutation frequency for the clustered DHT + 8-oxoG lesions than that for either a single 8-oxoG or a single DHT in wild-type and in glycosylase-deficient strains of ${it E. coli}$. From these results and similarities with the mutability of respective 8-oxoG + AP clusters, it is suggested that removal of 8-oxoG within clustered damage site is retarded, probably reflecting the preferential excision of DHT initially. For a certain fraction of clusters, 8-oxoG may be initially removed from the cluster. To gain further insights on the processing of the DHT + 8-oxoG cluster, several potential intermediates after 8-oxoG removal were assessed for their mutability. For instance, DHT + AP or DHT + Gap containing cluster, but not AP + AP or Gap +AP clusters, has a relatively low mutation frequency. Further, AP + AP or Gap + AP cluster had a reduced transformation efficiency. These results led us to suggest that, when either 8-oxoG or DHT is initially excised from a cluster containing 8-oxoG and DHT, the base remaining within the resulting damage will not be further converted to an AP site or to a single strand break ${it in vivo}$.

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