Development of the evaluation system for mutagenesis using DNA repair-deficient mice

由氧自由基引起的DNA或前体池内核苷酸的氧化损伤被认为在自发突变、致癌和衰老中起重要作用。特别是，8-oxodGTP和2-OHdATP是DNA合成的有效诱变底物。哺乳动物MTH1可以催化这些诱变底物的水解，这表明它可以防止这些氧化核苷酸引起的诱变。我们已经建立了缺乏8-oxodGTPase活性的MTH1^<-/->小鼠，这些小鼠被证明易患肺癌、肝癌和胃癌。为了研究由于MTH1缺乏引起的体内突变事件，将大肠杆菌的报告基因rpsL引入MTH1^<-/->小鼠。有趣的是，与MTH1^<+/+>小鼠相比，MTH1^<-/->小鼠中rpsL^-前向突变体的净频率没有明显增加。然而，我们发现这两种基因型在从mic中恢复的rpsL^-突变的类别和位点分布上存在差异。更多e.与mutt缺陷大肠杆菌的A:T→C:G转换频率比野生型细胞高1000倍不同，MTH1失合小鼠的A:T→C:G转换频率的增加并不明显。然而，MTH1^<-/->小鼠脾脏单核苷酸序列的单碱基帧移频率比野生型小鼠高5.7倍。由于单核苷酸运行时单碱基移的发生率升高是msh2依赖性错配修复系统缺陷的标志，因此MTH1^<-/->小鼠的这种弱位点特异性突变效应可归因于错配修复功能的部分隔离，该功能可能用于纠正与氧化核苷酸的错配。与这一假设相一致，MTH1^<-/-> MSH2^<-/->小鼠比单独MSH2^<-/->小鼠观察到G:C→T: a转换的频率显著增加。这些结果表明可能涉及多种抗诱变途径，包括MTH1蛋白和其他修复系统，在由氧化核苷酸引起的诱变中。少

Oxidative damage of nucleotides within DNA or precursor pools caused by oxygen radicals is thought to play an important role in spontaneous mutagenesis, as well as carcinogenesis and ageing. In particular, 8-oxodGTP and 2-OHdATP are potent mutagenic substrate for DNA synthesis. Mammalian MTH1 catalyzes hydrolysis of these mutagenic substrates, suggesting that it functions to prevent mutagenesis caused by these oxidized nucleotides. We have established MTH1^<-/-> mice lacking the 8-oxodGTPase activity, which were shown to be susceptible to lung, liver and stomach cancers. To examine in vivo mutation events due to the MTH1-deficiency, a reporter gene, rpsL of Escherichta coli, was introduced into MTH1^<-/-> mice. Interestingly, the net frequency of rpsL^- forward mutants showed no apparent increase in MTH1^<-/-> mice as compared to MTH1^<+/+> mice. However, we found differences between these two genotypes in the class- and site-distributions of the rpsL^- mutations recovered from the mic … More e. Unlike MutT-deficient E. coli showing 1,000-fold higher frequency of A:T→C:G transversions than the wild type cells, an increase in frequency of A:T→C:G transversion was not evident in MTH1 nullizygous mice. Nevertheless, the frequency of single-base frameshifts at mononucleotide runs was 5.7-fold higher in spleens of MTH1^<-/-> mice than in those of wild type mice. Since the elevated incidence of single-base frameshifts at mononucleotide runs is a hallmark of the defect in MSH2-dependent mismatch repair system, this weak site-specific mutator effect of MTH1^<-/-> mice could be attributed to a partial sequestration of the mismatch repair function that may act to correct mispairs with the oxidized nucleotides. Consistent with this hypothesis, a significant increase in the frequency of G:C→T:A transversions was observed with MTH1^<-/-> MSH2^<-/-> mice over MSH2^<-/-> mice alone. These results suggest a possible involvement of multiple anti-mutagenic pathways, including the MTH1 protein and other repair system(s), in mutagenesis caused by the oxdized nucleotides. Less