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Establishment of a sensitive assay system for detecting mutations induced by oxidative DNA damage

建立检测氧化DNA损伤诱导突变的灵敏检测系统

基本信息

批准号：
16310043
负责人：
TSUZUKI Teruhisa
金额：
$ 10.11万
依托单位：
KYUSHU UNIVERCITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

项目摘要

Oxygen radicals are produced through normal cellular metabolism, and formation of such radicals is enhanced further by ionizing radiation and by various chemicals. The oxygen radicals attack nucleic acids and generate various modified bases in DNA. Among them, 8-oxoguanine (8-oxoG) is the most abundant, and seems to play a critical role in mutagenesis as well as carcinogenesis and aging. 8-OxoG can pair with both cytosine and adenine during DNA synthesis, and as a result, G : C to T : A transversions are induced. Oxidation of guanine also occurs in the cellular nucleotide pool. 8-Oxo-dGTP, when formed, is a potent mutagenic substrate for DNA synthesis ; it is equally incorporated opposite both adenine and cytosine in DNA, resulting in both A : T to C : G and G : C to T : A transversions. In order to establish a sensitive assay system for detecting mutagenesis caused by oxidative DNA damage, we used the rpsL transgene as a reporter for the mutation assay, a suitable system for identifying a spectrum of base substitutions and single-base frameshifts. Using the resulting DNA repair-deficient mice with the rpsL transgene (rpsL hemizygotes), we examined oxidative stress-induced mutations occurring in the tissues of different DNA repair-deficient mouse lines. The frequency of G : C to T : A transversions was significantly increased both in Ogg1^<-/-> and Mutyh^<-/-> mice when administered with KBrO_3 in drinking water. We observed an increased incidence of G : C to A : T transitions in Ogg1^<-/-> mice after exposure to low dose X-ray irradiation (4 Gy). We further detected a characteristic spectra of A : T to G : C transitions in the spleens of X-ray-irradiated Mth1^<-/-> mice. In this study, we have shown a characteristic difference in the mutational spectra of wild-type mice and several DNA repair-deficient mice, such as Mth1,Ogg1,and Mutyh.We also examined the interaction between mammalian enzymes and oxidatively damaged ribonucleotides/RNA.

氧自由基是通过正常的细胞代谢产生的，电离辐射和各种化学物质进一步增强了这种自由基的形成。氧自由基攻击核酸并在DNA中产生各种修饰的碱基。其中，8-氧代鸟嘌呤（8-oxoG）是最丰富的，似乎在诱变以及致癌和衰老中发挥关键作用。8-在DNA合成过程中，OxoG可以与胞嘧啶和腺嘌呤配对，结果诱导G：C到T：A的颠换。鸟嘌呤的氧化也发生在细胞核苷酸库中。8-氧代-dGTP在形成时是DNA合成的有效诱变底物;它在DNA中与腺嘌呤和胞嘧啶相对地同等掺入，导致A：T至C：G和G：C至T：A颠换。为了建立一个敏感的检测系统，用于检测由氧化性DNA损伤引起的诱变，我们使用的rpsL转基因作为报告的突变试验，一个合适的系统，用于确定光谱的碱基取代和单碱基移码。使用所得的DNA修复缺陷小鼠与rpsL转基因（rpsL半合子），我们研究了氧化应激诱导的突变发生在不同的DNA修复缺陷小鼠系的组织。G的频率：C到T：饮水中给予KBrO_3后，Ogg ^<-/->和Mutyh^<-/->小鼠的A颠换均显著增加。我们观察到Ogg 1 ^<-/->小鼠暴露于低剂量X射线照射（4戈伊）后G：C至A：T转变的发生率增加。我们进一步在X射线照射的Mth 1 ^<-/->小鼠的脾脏中检测到A：T到G：C跃迁的特征光谱。在这项研究中，我们已经显示了野生型小鼠和几个DNA修复缺陷小鼠，如Mth 1，Ogg 1和Mutyh的突变谱的特征差异。我们还研究了哺乳动物酶和氧化损伤的核糖核苷酸/RNA之间的相互作用。

项目成果

期刊论文数量（39）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Two modes of microsatellite instability in human cancer: differential connection of defective DNA mismatch repair to dinucleotide repeat instability.

人类癌症中微卫星不稳定性的两种模式：有缺陷的DNA不匹配修复与二核苷酸重复不稳定性的差异连接。

DOI：
10.1093/nar/gki303
发表时间：
2005
期刊：
NUCLEIC ACIDS RESEARCH
影响因子：
14.9
作者：
Oda, S;Maehara, Y;Ikeda, Y;Oki, E;Egashira, A;Okamura, Y;Takahashi, I;Kakeji, Y;Sumiyoshi, Y;Miyashita, K;Yamada, Y;Zhao, Y;Hattori, H;Taguchi, K;Ikeuchi, T;Tsuzuki, T;Sekiguchi, M;Karran, P;Yoshida, MA
通讯作者：
Yoshida, MA

モデル動物の作製と維持(第12章 DNA修復酵素遺伝子-2を分担執筆)

模型动物的创建与维护（合着第12章DNA修复酶基因-2）