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

氧自由基引起的DNA或前体体内核苷酸的氧化损伤被认为在自发突变、癌症发生和衰老中发挥了重要作用。特别是8-oxodGTP和2-OHdATP是DNA合成的强诱变底物。哺乳动物的MTH1催化这些突变底物的水解，表明它具有防止这些氧化核苷酸引起的突变的功能。我们已经建立了MTH1^&lt；-/-&gt；缺乏8-oxodGTP酶活性的小鼠，这些小鼠被证明对肺癌、肝癌和胃癌易感。为了检测由于MTH1缺乏而导致的体内突变事件，将大肠杆菌的报告基因rpsL导入MTH1^-lt；-/-&gt；小鼠。有趣的是，与mth1^&lt；+/+&gt；小鼠相比，mth1^&lt；-/-&gt；小鼠rpsL^-Forward突变的净频率没有明显增加。然而，我们发现这两种基因型在从小鼠…中恢复的rpsL^-突变的类别和位置分布上存在差异。与缺乏突变的大肠杆菌不同，A：T→C：G颠换频率是野生型细胞的1,000倍，而在MTH1零合子小鼠中，A：T→C：G颠倒频率的增加并不明显。然而，在单核苷酸运行中，MTH1；-/-&gt；小鼠的脾中单碱基移码的频率是野生型小鼠的5.7倍。由于在单核苷酸运行时单碱基移码的发生率增加是MSH2依赖的错配修复系统缺陷的标志，MTH1^-/-&gt；小鼠的这种微弱的位点特异性突变效应可能归因于错配修复功能的部分隔离，该功能可能起到纠正与氧化核苷酸的错配的作用。与这一假设一致，观察到MTH1^&lt；-/-&gt；msh2^&gt；-/-&gt；小鼠的G：C→T：A易位频率显著高于msh2^&lt；-/-&gt；小鼠。这些结果表明，氧化核苷酸引起的突变可能涉及多条抗突变途径，包括MTH1蛋白和其他修复系统(S)。较少

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