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

氧自由基是通过正常的细胞代谢产生的，这种自由基的形成通过电离辐射和各种化学物质进一步增强。氧自由基攻击核酸并在DNA中产生各种修饰碱基。其中，8-氧鸟嘌呤（8-oxoG）含量最多，在突变、致癌、衰老等过程中发挥关键作用。在DNA合成过程中，8-OxoG可以与胞嘧啶和腺嘌呤配对，从而诱导G: C到T: a的转化。鸟嘌呤的氧化也发生在细胞核苷酸池中。8-Oxo-dGTP形成后，是DNA合成的有效诱变底物；它同样与DNA中的腺嘌呤和胞嘧啶结合，导致A: T到C: G和G: C到T: A的转化。为了建立一种灵敏的检测系统来检测氧化DNA损伤引起的突变，我们使用rpsL转基因作为突变检测的报告基因，这是一种适合识别碱基置换和单碱基移谱的系统。利用携带rpsL转基因的DNA修复缺陷小鼠（rpsL半合子），我们检测了不同DNA修复缺陷小鼠品系组织中发生的氧化应激诱导突变。饮水中添加KBrO_3后，Ogg1^<-/->和Mutyh^<-/->小鼠G: C向T: A转化的频率均显著增加。我们观察到Ogg1^<-/->小鼠在低剂量x射线照射（4gy）后G: C向A: T转变的发生率增加。我们进一步检测了x射线照射Mth1^<-/->小鼠脾脏中a: T到G: C转变的特征光谱。在本研究中，我们发现野生型小鼠和几种DNA修复缺陷小鼠（如Mth1、Ogg1和Mutyh）的突变谱存在特征性差异。我们还研究了哺乳动物酶与氧化损伤核糖核苷酸/RNA之间的相互作用。

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.