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新陈代谢产生的，电离辐射和各种化学物质进一步促进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-氧鸟嘌呤(8-oxoG)含量最丰富，似乎在诱变、致癌和衰老过程中起着关键作用。在DNA合成过程中，8-OxoG既能与胞嘧啶配对，又能与腺嘌呤配对，导致G：C到T：A的转换。鸟嘌呤的氧化也发生在细胞的核苷酸池中。8-oxo-dGTP一旦形成，就是DNA合成的有效诱变底物；它在DNA中与腺嘌呤和胞嘧啶相反地掺入，导致A：T到C：G和G：C到T：A的颠换。为了建立检测DNA氧化损伤所致突变的灵敏检测体系，我们使用rpsL转基因作为突变检测的报告基因，这是一种适合于鉴定一系列碱基替换和单碱基移码的系统。使用rpsL转基因DNA修复缺陷小鼠(rpsL半合子)，我们检测了氧化应激诱导的突变发生在不同DNA修复缺陷小鼠的组织中。饮水中加入KBrO_3后，Ogg1^&lt；-/-&gt；和MUTYH^&lt；-/-&gt；小鼠的G：C到T：A易位频率均显著增加。我们观察到Ogg1^&lt；-/-&gt；小鼠暴露在低剂量X射线照射(4Gy射线)后，G：C到A：T转变的发生率增加。我们进一步在X射线照射的小鼠的脾中检测到了A：T到G：C跃迁的特征光谱。在这项研究中，我们显示了野生型小鼠和几种DNA修复缺陷小鼠，如MTH1，Ogg1和Mutyh的突变谱的特征差异。我们还研究了哺乳动物酶与氧化损伤的核苷酸/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.